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Sample records for chaperone asf1 levels

  1. Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

    PubMed Central

    Tanae, Katsuhiro; Horiuchi, Tomitaka; Matsuo, Yuzy; Katayama, Satoshi; Kawamukai, Makoto

    2012-01-01

    The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe. PMID:22291963

  2. Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei

    PubMed Central

    Pascoalino, Bruno; Dindar, Gülcin; Vieira-da-Rocha, João P.; Machado, Carlos Renato; Janzen, Christian J.; Schenkman, Sergio

    2014-01-01

    The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes. PMID:24322299

  3. Loss of the histone chaperone ASF1B reduces female reproductive capacity in mice.

    PubMed

    Messiaen, S; Guiard, J; Aigueperse, C; Fliniaux, I; Tourpin, S; Barroca, V; Allemand, I; Fouchet, P; Livera, G; Vernet, M

    2016-05-01

    Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1b(GT(ROSA-βgeo)437)) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of β-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process. PMID:26850882

  4. Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery.

    PubMed

    Moshkin, Yuri M; Armstrong, Jennifer A; Maeda, Robert K; Tamkun, John W; Verrijzer, Peter; Kennison, James A; Karch, Francois

    2002-10-15

    De novo chromatin assembly into regularly spaced nucleosomal arrays is essential for eukaryotic genome maintenance and inheritance. The Anti-Silencing Function 1 protein (ASF1) has been shown to be a histone chaperone, participating in DNA-replication-coupled nucleosome assembly. We show that mutations in the Drosophila asf1 gene derepress silencing at heterochromatin and that the ASF1 protein has a cell cycle-specific nuclear and cytoplasmic localization. Furthermore, using both genetic and biochemical methods, we demonstrate that ASF1 interacts with the Brahma (SWI/SNF) chromatin-remodelling complex. These findings suggest that ASF1 plays a crucial role in both chromatin assembly and SWI/SNF-mediated chromatin remodelling. PMID:12381660

  5. Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function.

    PubMed

    Seol, Ja-Hwan; Song, Tae-Yang; Oh, Se Eun; Jo, Chanhee; Choi, Ahreum; Kim, Byungho; Park, Jinyoung; Hong, Suji; Song, Ilrang; Jung, Kwan Young; Yang, Jae-Hyun; Park, Hwangseo; Ahn, Jin-Hyun; Han, Jeung-Whan; Cho, Eun-Jung

    2015-12-01

    The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modification. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. PMID:26058396

  6. Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1

    PubMed Central

    O’Sullivan, Roderick J.; Arnoult, Nausica; Lackner, Daniel H.; Oganesian, Liana; Haggblom, Candy; Corpet, Armelle; Almouzni, Genevieve; Karlseder, Jan

    2013-01-01

    The mechanism of activation of the Alternative Lengthening of Telomeres (ALT) pathway of mammalian chromosome end maintenance has remained an unresolved issue. We have discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT associated PML bodies (APBs), extra-chromosomal telomeric DNA species an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and inter-telomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We identified that ALT induction is positively regulated by RAD17 and BLM, while negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly support the hypothesis that ALT is a consequence of a histone management dysfunction. PMID:24413054

  7. Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation.

    PubMed

    Das, Chandrima; Roy, Siddhartha; Namjoshi, Sarita; Malarkey, Christopher S; Jones, David N M; Kutateladze, Tatiana G; Churchill, Mair E A; Tyler, Jessica K

    2014-03-25

    The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. Here we demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. We show that autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and we propose that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP. PMID:24616510

  8. Cell reprogramming. Histone chaperone ASF1A is required for maintenance of pluripotency and cellular reprogramming.

    PubMed

    Gonzalez-Muñoz, Elena; Arboleda-Estudillo, Yohanna; Otu, Hasan H; Cibelli, Jose B

    2014-08-15

    Unfertilized oocytes have the intrinsic capacity to remodel sperm and the nuclei of somatic cells. The discoveries that cells can change their phenotype from differentiated to embryonic state using oocytes or specific transcription factors have been recognized as two major breakthroughs in the biomedical field. Here, we show that ASF1A, a histone-remodeling chaperone specifically enriched in the metaphase II human oocyte, is necessary for reprogramming of human adult dermal fibroblasts (hADFs) into undifferentiated induced pluripotent stem cell. We also show that overexpression of just ASF1A and OCT4 in hADFs exposed to the oocyte-specific paracrine growth factor GDF9 can reprogram hADFs into pluripotent cells. Our Report underscores the importance of studying the unfertilized MII oocyte as a means to understand the molecular pathways governing somatic cell reprogramming. PMID:25035411

  9. Structure of a Human ASF1a-HIRA Complex and Insights into Specificity of Histone Chaperone Complex Assembly

    SciTech Connect

    Tang,Y.; Poustovoitov, M.; Zhao, K.; Garfinkel, M.; Canutescu, A.; Dunbrack, R.; Adams, P.; Marmorstein, R.

    2006-01-01

    Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin-assembly complexes, with roles liked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a-HIRA heterodimer and a biochemical dissection of ASFA1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B domain forms an antiparallel {beta}-hairpin that binds perpendicular to the strands of the {beta}-sandwich of ASF1a, via {beta}-sheet, salt bridge and van der Waals contacts. the N- and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B domain. CAF-1 p60 also uses B domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes.

  10. The C Terminus of the Histone Chaperone Asf1 Cross-Links to Histone H3 in Yeast and Promotes Interaction with Histones H3 and H4

    PubMed Central

    Dennehey, Briana K.; Noone, Seth; Liu, Wallace H.; Smith, Luke

    2013-01-01

    The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing. PMID:23184661

  11. Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing.

    PubMed

    Moshkin, Yuri M; Kan, Tsung Wai; Goodfellow, Henry; Bezstarosti, Karel; Maeda, Robert K; Pilyugin, Maxim; Karch, Francois; Bray, Sarah J; Demmers, Jeroen A A; Verrijzer, C Peter

    2009-09-24

    Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing. PMID:19782028

  12. Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer

    PubMed Central

    Corpet, Armelle; De Koning, Leanne; Toedling, Joern; Savignoni, Alexia; Berger, Frédérique; Lemaître, Charlène; O'Sullivan, Roderick J; Karlseder, Jan; Barillot, Emmanuel; Asselain, Bernard; Sastre-Garau, Xavier; Almouzni, Geneviève

    2011-01-01

    Mammalian cells possess two isoforms of the histone H3–H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis. PMID:21179005

  13. Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer.

    PubMed

    Corpet, Armelle; De Koning, Leanne; Toedling, Joern; Savignoni, Alexia; Berger, Frédérique; Lemaître, Charlène; O'Sullivan, Roderick J; Karlseder, Jan; Barillot, Emmanuel; Asselain, Bernard; Sastre-Garau, Xavier; Almouzni, Geneviève

    2011-02-01

    Mammalian cells possess two isoforms of the histone H3-H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis. PMID:21179005

  14. The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4–DNA complexes

    PubMed Central

    Donham, Douglas C.; Scorgie, Jean K.; Churchill, Mair E. A.

    2011-01-01

    The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin. PMID:21447559

  15. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

  16. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    NASA Astrophysics Data System (ADS)

    Klimovskaia, Ilnaz M.; Young, Clifford; Strømme, Caroline B.; Menard, Patrice; Jasencakova, Zuzana; Mejlvang, Jakob; Ask, Katrine; Ploug, Michael; Nielsen, Michael L.; Jensen, Ole N.; Groth, Anja

    2014-03-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

  17. Roles of p53 and ASF1A in the Reprogramming of Sheep Kidney Cells to Pluripotent Cells.

    PubMed

    Shi, Huijun; Fu, Qiang; Li, Guozhong; Ren, Yan; Hu, Shengwei; Ni, Wei; Guo, Fei; Shi, Mengting; Meng, Luping; Zhang, Hui; Qiao, Jun; Guo, Zhiru; Chen, Chuangfu

    2015-12-01

    Since the first report of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka, numerous attempts have been made to derive iPSCs from other species via the ectopic expression of defined factors. Sheep iPSCs (siPSCs) have significant potential for biotechnology and agriculture. Although several groups have described siPSCs, the reprogramming efficiency was extremely low. The exogenous transgenes could be not silenced in the iPSCs, which hampered their development and application. Here, we report that p53 knockdown and antisilencing function 1A (ASF1A) overexpression promoted iPSC generation from sheep kidney cells (SKCs). Compared with transduction with eight human defined transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, hTERT, and SV40LT), the additional introduction of p53 RNA interference (RNAi) and/or ASF1A in the presence of small-molecule compounds [vitamin C (Vc) and valproic acid (VPA)] greatly improved the efficiency of sheep iPSC generation. The siPSCs exhibited morphological features similar to mouse embryonic stem cells (ESCs) and were positive for alkaline phosphatase and, pluripotent marker genes (Oct4, Nanog, Sox2, Rex1, TRA-1-60, TRA-1-81, and E-cadherin). Furthermore, these cells exhibited a normal karyotype of 54 chromosomes and were able to differentiate into all three germ layers both in vitro and in vivo. Moreover, the exogenous genes were silenced in siPSCs when p53 small hairpin RNA (shRNA) and ASF1A were added. Our results may help to reveal the role of p53 and ASF1A in sheep somatic cell reprogramming and provide an efficient approach to reprogramming sheep somatic cells. PMID:26580119

  18. DNA Mismatch Repair Interacts with CAF-1- and ASF1A-H3-H4-dependent Histone (H3-H4)2 Tetramer Deposition.

    PubMed

    Rodriges Blanko, Elena; Kadyrova, Lyudmila Y; Kadyrov, Farid A

    2016-04-22

    DNA mismatch repair (MMR) is required for the maintenance of genome stability and protection of humans from several types of cancer. Human MMR occurs in the chromatin environment, but little is known about the interactions between MMR and the chromatin environment. Previous research has suggested that MMR coincides with replication-coupled assembly of the newly synthesized DNA into nucleosomes. The first step in replication-coupled nucleosome assembly is CAF-1-dependent histone (H3-H4)2 tetramer deposition, a process that involves ASF1A-H3-H4 complex. In this work we used reconstituted human systems to investigate interactions between MMR and CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition. We have found that MutSα inhibits CAF-1- and ASF1A-H3-H4-dependent packaging of a DNA mismatch into a tetrasome. This finding supports the idea that MMR occurs before the DNA mismatch is packaged into the tetrasome. Our experiments have also revealed that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers does not interfere with MMR reactions. In addition, we have established that unnecessary degradation of the discontinuous strand that takes place in both DNA polymerase δ (Pol δ)- and DNA polymerase ϵ (Pol ϵ)-dependent MMR reactions is suppressed by CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers. These data suggest that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers is compatible with MMR and protects the discontinuous daughter strand from unnecessary degradation by MMR machinery. PMID:26945061

  19. Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level

    PubMed Central

    Takeuchi, Toshihide; Suzuki, Mari; Fujikake, Nobuhiro; Popiel, H. Akiko; Kikuchi, Hisae; Futaki, Shiroh; Wada, Keiji; Nagai, Yoshitaka

    2015-01-01

    The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non–cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis. PMID:25918398

  20. High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli.

    PubMed

    Mahamad, Pornpimol; Boonchird, Chuenchit; Panbangred, Watanalai

    2016-07-01

    CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 ± 8.95 μg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 μg/mL and 150.07 ± 8.13 μg/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host. PMID:27020286

  1. ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

    PubMed Central

    Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L.; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

    2013-01-01

    ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra. PMID:23447699

  2. Targeting the prodromal stage of spinocerebellar ataxia type 17 mice: G-CSF in the prevention of motor deficits via upregulating chaperone and autophagy levels.

    PubMed

    Chang, Ya-Chin; Lin, Chia-Wei; Hsu, Chen-Ming; Lee-Chen, Guey-Jen; Su, Ming-Tsan; Ro, Long-Sun; Chen, Chiung-Mei; Huang, Hei-Jen; Hsieh-Li, Hsiu Mei

    2016-05-15

    Spinocerebellar ataxia type 17 (SCA17), an autosomal dominant cerebellar ataxia, is a devastating, incurable disease caused by the polyglutamine (polyQ) expansion of transcription factor TATA binding protein (TBP). The polyQ expansion causes misfolding and aggregation of the mutant TBP, further leading to cytotoxicity and cell death. The well-recognized prodromal phase in many forms of neurodegeneration suggests a prolonged period of partial neuronal dysfunction prior to cell loss that may be amenable to therapeutic intervention. The objective of this study was to assess the effects and molecular mechanisms of granulocyte-colony stimulating factor (G-CSF) therapy during the pre-symptomatic stage in SCA17 mice. Treatment with G-CSF at the pre-symptomatic stage improved the motor coordination of SCA17 mice and reduced the cell loss, insoluble mutant TBP protein, and vacuole formation in the Purkinje neurons of these mice. The neuroprotective effects of G-CSF may be produced by increases in Hsp70, Beclin-1, LC3-II and the p-ERK survival pathway. Upregulation of chaperone and autophagy levels further enhances the clearance of mutant protein aggregation, slowing the progression of pathology in SCA17 mice. Therefore, we showed that the early intervention of G-CSF has a neuroprotective effect, delaying the progression of SCA17 in mutant mice via increases in the levels of chaperone expression and autophagy. PMID:26972528

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

    SciTech Connect

    Umehara, Takashi; Otta, Yumi; Tsuganezawa, Keiko; Matsumoto, Takehisa; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2005-11-01

    The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1{sup +} is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1{sup +}-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation.

  4. Gymnastics of molecular chaperones.

    PubMed

    Mayer, Matthias P

    2010-08-13

    Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families. PMID:20705236

  5. Forces Driving Chaperone Action.

    PubMed

    Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C A

    2016-07-14

    It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client's affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

  6. Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex

    PubMed Central

    Daniel Ricketts, M; Frederick, Brian; Hoff, Henry; Tang, Yong; Schultz, David C.; Singh Rai, Taranjit; Grazia Vizioli, Maria; Adams, Peter D.; Marmorstein, Ronen

    2015-01-01

    Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1–H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex. PMID:26159857

  7. Chaperones in Neurodegeneration

    PubMed Central

    Shorter, James; Wiseman, R. Luke; Chiti, Fabrizio; Dickey, Chad A.; McLean, Pamela J.

    2015-01-01

    Cellular protein homeostasis (proteostasis) maintains the integrity of the proteome and includes protein synthesis, folding, oligomerization, and turnover; chaperone proteins assist with all of these processes. Neurons appear to be especially susceptible to failures in proteostasis, and this is now increasingly recognized as a major origin of neurodegenerative disease. This review, based on a mini-symposium presented at the 2015 Society for Neuroscience meeting, describes new work in the area of neuronal proteostasis, with a specific focus on the roles and therapeutic uses of protein chaperones. We first present a brief review of protein misfolding and aggregation in neurodegenerative disease. We then discuss different aspects of chaperone control of neuronal proteostasis on topics ranging from chaperone engineering, to chaperone-mediated blockade of protein oligomerization and cytotoxicity, to the potential rescue of neurodegenerative processes using modified chaperone proteins. SIGNIFICANCE STATEMENT Aberrant protein homeostasis within neurons results in protein misfolding and aggregation. In this review, we discuss specific roles for protein chaperones in the oligomerization, assembly, and disaggregation of proteins known to be abnormally folded in neurodegenerative disease. Collectively, our goal is to identify therapeutic mechanisms to reduce the cellular toxicity of abnormal aggregates. PMID:26468185

  8. Structural mechanisms of chaperone mediated protein disaggregation

    PubMed Central

    Sousa, Rui

    2014-01-01

    The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID

  9. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  10. High levels of Hsp90 co-chaperone p23 promotes tumor progression and poor prognosis in breast cancer by increasing lymph node metastases and drug resistance

    PubMed Central

    Simpson, Natalie E.; Lambert, W. Marcus; Watkins, Renecia; Giashuddin, Shah; Huang, S. Joseph; Oxelmark, Ellinor; Arju, Rezina; Hochman, Tsivia; Goldberg, Judith D.; Schneider, Robert J.; Reiz, Luiz Fernando Lima; Soares, Fernando Augusto; Logan, Susan K.; Garabedian, Michael J.

    2010-01-01

    p23 is an Hsp90 co-chaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls catalytic activity of certain kinases, regulates protein-DNA dynamics and is upregulated in several cancers. We previously demonstrated that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients. PMID:20847343

  11. Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation.

    PubMed

    Qiu, Hongfang; Chereji, Răzvan V; Hu, Cuihua; Cole, Hope A; Rawal, Yashpal; Clark, David J; Hinnebusch, Alan G

    2016-02-01

    Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes. PMID:26602697

  12. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

    PubMed

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication. PMID:26167883

  13. Molecular chaperones and neuronal proteostasis

    PubMed Central

    Smith, Heather L.; Li, Wenwen; Cheetham, Michael E.

    2015-01-01

    Protein homeostasis (proteostasis) is essential for maintaining the functionality of the proteome. The disruption of proteostasis, due to genetic mutations or an age-related decline, leads to aberrantly folded proteins that typically lose their function. The accumulation of misfolded and aggregated protein is also cytotoxic and has been implicated in the pathogenesis of neurodegenerative diseases. Neurons have developed an intrinsic protein quality control network, of which molecular chaperones are an essential component. Molecular chaperones function to promote efficient folding and target misfolded proteins for refolding or degradation. Increasing molecular chaperone expression can suppress protein aggregation and toxicity in numerous models of neurodegenerative disease; therefore, molecular chaperones are considered exciting therapeutic targets. Furthermore, mutations in several chaperones cause inherited neurodegenerative diseases. In this review, we focus on the importance of molecular chaperones in neurodegenerative diseases, and discuss the advances in understanding their protective mechanisms. PMID:25770416

  14. Measurement and modification of the expression level of the chaperone protein and signaling regulator GRP78/BiP in mammalian cells.

    PubMed

    Chen, Wan-Ting; Lee, Amy S

    2011-01-01

    GRP78/BiP is a major endoplasmic reticulum (ER) chaperone protein essential for protein quality control in the ER as well as a central regulator of unfolded protein response (UPR). The induction of GRP78 is well established as a marker for ER stress. Recently, mouse models targeting the Grp78 allele indicate that GRP78 has critical roles in cancer progression, drug resistance, angiogenesis, neurological diseases, and diabetes. The discovery of a cytosolic GRP78 isoform and cell surface GRP78 adds new insights to its function beyond the ER compartment in regulating growth factor signaling and cell viability. Here, we summarize and update several approaches for the detection and quantitation of total GRP78, cytosolic GRP78 isoform, and cell surface GRP78, and the use of small interfering RNA to knockdown GRP78 expression. These techniques can be applied to culture cells as well as tissues. PMID:21266253

  15. Polyphosphate is a primordial chaperone.

    PubMed

    Gray, Michael J; Wholey, Wei-Yun; Wagner, Nico O; Cremers, Claudia M; Mueller-Schickert, Antje; Hock, Nathaniel T; Krieger, Adam G; Smith, Erica M; Bender, Robert A; Bardwell, James C A; Jakob, Ursula

    2014-03-01

    Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation. In vitro studies reveal that polyP has protein-like chaperone qualities, binds to unfolding proteins with high affinity in an ATP-independent manner, and supports their productive refolding once nonstress conditions are restored. Our results uncover a universally important function for polyP and suggest that these long chains of inorganic phosphate may have served as one of nature's first chaperones, a role that continues to the present day. PMID:24560923

  16. Do nucleic acids moonlight as molecular chaperones?

    PubMed Central

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  17. Do nucleic acids moonlight as molecular chaperones?

    PubMed

    Docter, Brianne E; Horowitz, Scott; Gray, Michael J; Jakob, Ursula; Bardwell, James C A

    2016-06-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  18. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  19. Molecular chaperones and the epigenetics of longevity and cancer resistance.

    PubMed

    Krøll, Jens

    2007-04-01

    The inherent immortality of embryonic stem cells demonstrates that replicative senescence as possibly the aging of species are epigenetic phenomena. The cellular level of expression of the housekeeping molecular chaperones correlates with longevity and cancer resistance of species. The chaperones are cancer antagonists by acting as genetic buffers, stabilizing the normal phenotype. Probably the progressive age-related silencing of the housekeeping genes contributes to the phenotype of aging, with the associated increase in cancer incidence. The present review concerns epigenetic chemical, immunological, and hormonal mechanisms, activating chaperone- and immune-response genes, which have proved effective in increasing longevity and cancer resistance. The relation of steroid hormone levels to species longevity, the anticarcinogenic activity of pregnancy hormones, and the influence of hormones on the longevity of social insects, illustrates the importance of hormonal mechanisms for the activation of longevity genes. PMID:17460166

  20. Altered mRNA Levels of Glucocorticoid Receptor, Mineralocorticoid Receptor, and Co-Chaperones (FKBP5 and PTGES3) in the Middle Frontal Gyrus of Autism Spectrum Disorder Subjects.

    PubMed

    Patel, Neil; Crider, Amanda; Pandya, Chirayu D; Ahmed, Anthony O; Pillai, Anilkumar

    2016-05-01

    Although stress has been implicated in the pathophysiology of autistic spectrum disorder (ASD), it is not known whether glucocorticoid receptor (GR) levels are altered in the brain of subjects with ASD. The messenger RNA (mRNA) levels of GR isoforms (GRα, GRβ, GRγ, and GRP), mineralocorticoid receptor (MR), GR co-chaperones (FKBP5, PTGES3, and BAG1), and inflammatory cytokines (IL-6, IL-1β, and IFN-γ) were examined in the postmortem middle frontal gyrus tissues of 13 ASD and 13 age-matched controls by qRT-PCR. The protein levels were examined by Western blotting. We found significant decreases in GRα (64 %), GRγ (48 %), GRP (20 %) and MR (46 %) mRNA levels in ASD subjects as compared to controls. However, significant increases in FKBP5 (42 %) and PTGES3 (35 %) mRNA levels were observed in ASD subjects. There were no differences in the mRNA levels of GRβ and BAG1 in ASD subjects as compared to controls. MR mRNA was found to be negatively correlated with the diagnostic score for abnormality of development. On the protein level, significant reductions in GR and MR, but no change in FKBP5 and PTGES3 were found in ASD subjects as compared to controls. Moreover, we observed significant increases in IL-1β and IFN-γ mRNA levels in ASD subjects, and these cytokines were negatively associated with GR levels. Our data, for the first time, reports dysregulation of GR, MR, FKBP5, and PTGES3 in ASD and suggest a possible role of inflammation in altered GR function in ASD. PMID:25912394

  1. RUBISCO ACTIVASE --- RUBISCO'S CATALYTIC CHAPERONE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The current status of research on the structure, regulation, mechanism and importance of Rubisco activase is reviewed. The activase is now recognized to be a member of the AAA+ family, whose members participate in macromolecular complexes that perform diverse chaperone-line functions. The conversed ...

  2. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

    PubMed

    Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

    2016-07-01

    A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis. PMID:27038828

  3. Copper transporters and chaperones: Their function on angiogenesis and cellular signalling.

    PubMed

    Bharathi Devi, S R; Dhivya M, Aloysius; Sulochana, K N

    2016-09-01

    Copper, although known as a micronutrient, has a pivotal role in modulating the cellular metabolism. Many studies have reported the role of copper in angiogenesis. Copper chaperones are intracellular proteins that mediate copper trafficking to various cell organelles. However, the role and function of copper chaperones in relation to angiogenesis has to be further explored. The intracellular copper levels when in excess are deleterious and certain mutations of copper chaperones have been shown to induce cell death and influence various cellular metabolisms. The study of these chaperones will be helpful in understanding the players in the cascade of events in angiogenesis and their role in cellular metabolic pathways. In this review we have briefly listed the copper chaperones associated with angiogenic and metabolic signalling and their function. PMID:27581939

  4. Pharmacological Targeting of the Hsp70 Chaperone

    PubMed Central

    Patury, Srikanth; Miyata, Yoshinari; Gestwicki, Jason E.

    2009-01-01

    The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein’s life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery. PMID:19860737

  5. Endoplasmic Reticulum Chaperones and Their Roles in the Immunogenicity of Cancer Vaccines

    PubMed Central

    Graner, Michael W.; Lillehei, Kevin O.; Katsanis, Emmanuel

    2015-01-01

    The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called “chaperone-rich cell lysate” (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes. PMID:25610811

  6. Molecular chaperones and proteostasis regulation during redox imbalance☆

    PubMed Central

    Niforou, Katerina; Cheimonidou, Christina; Trougakos, Ioannis P.

    2014-01-01

    Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones. PMID:24563850

  7. Visualizing chaperone-assisted protein folding.

    PubMed

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S; Martin, Raoul; Quan, Shu; Afonine, Pavel V; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C; Brooks, Charles L; Bardwell, James C A

    2016-07-01

    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone-substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone. PMID:27239796

  8. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy

    PubMed Central

    Quan, Shu; Koldewey, Philipp; Tapley, Tim; Kirsch, Nadine; Ruane, Karen M.; Pfizenmaier, Jennifer; Shi, Rong; Hofmann, Stephan; Foit, Linda; Ren, Guoping; Jakob, Ursula; Xu, Zhaohui; Cygler, Miroslaw; Bardwell, James C. A.

    2011-01-01

    To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. Spy is unlike the structure of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors. PMID:21317898

  9. Localization of the chaperone binding site

    NASA Technical Reports Server (NTRS)

    Boyle, D.; Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

  10. Threonine 22 phosphorylation attenuates Hsp90 interaction with co-chaperones and affects its chaperone activity

    PubMed Central

    Mollapour, Mehdi; Tsutsumi, Shinji; Truman, Andrew W.; Xu, Wanping; Vaughan, Cara K.; Beebe, Kristin; Konstantinova, Anna; Vourganti, Srinivas; Panaretou, Barry; Piper, Peter W.; Trepel, Jane B.; Prodromou, Chrisostomos; Pearl, Laurence H.; Neckers, Len

    2011-01-01

    SUMMARY Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by co-chaperones but also by distinct post-translational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α-helix 1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α-helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase competent state. Phospho-mimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function, and impacts interaction with the co-chaperones Aha1 and Cdc37. Over-expression of Aha1 stimulates the ATPase activity, restores co-chaperone interactions, and compensates for the functional defects of these Hsp90 mutants. PMID:21419342

  11. Using pharmacological chaperones to restore proteostasis

    PubMed Central

    Wang, Ya-Juan; Di, Xiao-Jing; Mu, Ting-Wei

    2014-01-01

    Normal organismal physiology depends on the maintenance of proteostasis in each cellular compartment to achieve a delicate balance between protein synthesis, folding, trafficking, and degradation while minimizing misfolding and aggregation. Defective proteostasis leads to numerous protein misfolding diseases. Pharmacological chaperones are cell-permeant small molecules that promote the proper folding and trafficking of a protein via direct binding to that protein. They stabilize their target protein in a protein-pharmacological chaperone state, increasing the natively-folded protein population that can effectively engage trafficking machinery for transport to the final destination for function. Here, as regards the application of pharmacological chaperones, we focus on their capability to promote the folding and trafficking of lysosomal enzymes, G protein coupled receptors (GPCRs), and ion channels, each of which is presently an important drug target. Pharmacological chaperones hold great promise as potential therapeutics to ameliorate a variety of protein misfolding diseases. PMID:24747662

  12. Molecular chaperones: functional mechanisms and nanotechnological applications

    NASA Astrophysics Data System (ADS)

    Rosario Fernández-Fernández, M.; Sot, Begoña; María Valpuesta, José

    2016-08-01

    Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.

  13. Molecular chaperones: functional mechanisms and nanotechnological applications.

    PubMed

    Fernández-Fernández, M Rosario; Sot, Begoña; Valpuesta, José María

    2016-08-12

    Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures. PMID:27363314

  14. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    PubMed

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models. PMID:27415450

  15. A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation.

    PubMed

    Tripathi, Amit K; Pareek, Ashwani; Singla-Pareek, Sneh Lata

    2016-08-01

    Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants. PMID:27342307

  16. A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation1[OPEN

    PubMed Central

    Pareek, Ashwani; Singla-Pareek, Sneh Lata

    2016-01-01

    Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants. PMID:27342307

  17. Multitasking SecB chaperones in bacteria

    PubMed Central

    Sala, Ambre; Bordes, Patricia; Genevaux, Pierre

    2014-01-01

    Protein export in bacteria is facilitated by the canonical SecB chaperone, which binds to unfolded precursor proteins, maintains them in a translocation competent state and specifically cooperates with the translocase motor SecA to ensure their proper targeting to the Sec translocon at the cytoplasmic membrane. Besides its key contribution to the Sec pathway, SecB chaperone tasking is critical for the secretion of the Sec-independent heme-binding protein HasA and actively contributes to the cellular network of chaperones that control general proteostasis in Escherichia coli, as judged by the significant interplay found between SecB and the trigger factor, DnaK and GroEL chaperones. Although SecB is mainly a proteobacterial chaperone associated with the presence of an outer membrane and outer membrane proteins, secB-like genes are also found in Gram-positive bacteria as well as in certain phages and plasmids, thus suggesting alternative functions. In addition, a SecB-like protein is also present in the major human pathogen Mycobacterium tuberculosis where it specifically controls a stress-responsive toxin–antitoxin system. This review focuses on such very diverse chaperone functions of SecB, both in E. coli and in other unrelated bacteria. PMID:25538690

  18. Molecular chaperones: multiple functions, pathologies, and potential applications.

    PubMed

    Macario, Alberto J L; Conway de Macario, Everly

    2007-01-01

    Cell stressors are ubiquitous and frequent, challenging cells often, which leads to the stress response with activation of anti-stress mechanisms. These mechanisms involve a variety of molecules, including molecular chaperones also known as heat-shock proteins (Hsp). The chaperones treated in this article are proteins that assist other proteins to fold, refold, travel to their place of residence (cytosol, organelle, membrane, extracellular space), and translocate across membranes. Molecular chaperones participate in a variety of physiological processes and are widespread in organisms, tissues, and cells. It follows that chaperone failure will have an impact, possibly serious, on one or more cellular function, which may lead to disease. Chaperones must recognize and interact with proteins in need of assistance or client polypeptides (e.g., nascent at the ribosome, or partially denatured by stressors), and have to interact with other chaperones because the chaperoning mechanism involves teams of chaperone molecules, i.e., multimolecular assemblies or chaperone machines. Consequently, chaperone molecules have structural domains with distinctive functions: bind the client polypeptide, interact with other chaperone molecules to build a machine, and interact with other complexes that integrate the chaperoning network. Also, various chaperones have ATP-binding and ATPase sites because the chaperoning process requires as, a rule, energy from ATP hydrolysis. Alterations in any one of these domains due to a mutation or an aberrant post-translational modification can disrupt the chaperoning process and cause diseases termed chaperonopathies. This article presents the pathologic concept of chaperonopathy with examples, and discusses the potential of using chaperones (genes or proteins) in treatment (chaperonotherapy). In addition, emerging topics within the field of study of chaperones (chaperonology) are highlighted, e.g., genomics (chaperonomics), systems biology

  19. Novel interaction between the major bacterial heat shock chaperone (GroESL) and an RNA chaperone (CspC).

    PubMed

    Lenz, Gal; Ron, Eliora Z

    2014-01-23

    The heat shock response is one of the main global regulatory networks in all organisms and involves an increased cellular level of chaperones and proteases to enable correct protein folding and balanced growth. One of the major heat shock chaperones in Escherichia coli is GroESL, composed of GroES and GroEL (the bacterial Hsp10 and Hsp60 homologues), which is essential for refolding of misfolded proteins. GroESL was previously shown to play a role in the regulation of the heat shock response by promoting the proteolysis of the regulatory protein--sigma32 (RpoH), the heat shock transcription activator. Here we show the involvement of GroESL in another proteolytic process, this of the major RNA chaperone--CspC--that specifically stabilizes the transcripts of several stress-related genes. Evidence is provided for an interaction between GroESL and CspC that results in enhanced, temperature-dependent proteolysis of the latter. This interaction is of regulatory importance, as reduction in the cellular levels of CspC leads to a decrease in stability of the major heat shock gene transcripts. PMID:24148697

  20. Potential synergy between tau aggregation inhibitors and tau chaperone modulators

    PubMed Central

    2013-01-01

    Tau is a soluble, microtubule-associated protein known to aberrantly form amyloid-positive aggregates. This pathology is characteristic for more than 15 neuropathies, the most common of which is Alzheimer’s disease. Finding therapeutics to reverse or remove this non-native tau state is of great interest; however, at this time only one drug is entering phase III clinical trials for treating tauopathies. Generally, tau manipulation by therapeutics can either directly or indirectly alter tau aggregation and stability. Drugs that bind and change the conformation of tau itself are largely classified as aggregation inhibitors, while drugs that alter the activity of a tau-effector protein fall into several categories, such as kinase inhibitors, microtubule stabilizers, or chaperone modulators. Chaperone inhibitors that have proven effective in tau models include heat shock protein 90 inhibitors, heat shock protein 70 inhibitors and activators, as well as inducers of heat shock proteins. While many of these compounds can alter tau levels and/or aggregation states, it is possible that combining these approaches may produce the most optimal outcome. However, because many of these compounds have multiple off-target effects or poor blood–brain barrier permeability, the development of this synergistic therapeutic strategy presents significant challenges. This review will summarize many of the drugs that have been identified to alter tau biology, with special focus on therapeutics that prevent tau aggregation and regulate chaperone-mediated clearance of tau. PMID:24041111

  1. Pharmacological Chaperoning: A Primer on Mechanism and Pharmacology

    PubMed Central

    Ryder, Katelyn G.

    2014-01-01

    Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs is considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast

  2. Supercharging Chaperones: A Meeting Toolkit for Maximizing Learning for Youth and Chaperones

    ERIC Educational Resources Information Center

    Brandt, Brian

    2016-01-01

    Trip and conference chaperones are a wonderful resource in youth development programs. These well-intended volunteers, many parents of youth participating in the event, want the best experience for the youth but are not necessarily trained in positive youth development. A consequence of this circumstance is that not all chaperones provide the best…

  3. Aging cellular networks: chaperones as major participants.

    PubMed

    Soti, C; Csermely, P

    2007-01-01

    We increasingly rely on the network approach to understand the complexity of cellular functions. Chaperones (heat shock proteins) are key "networkers", which sequester and repair damaged proteins. In order to link the network approach and chaperones with the aging process, we first summarize the properties of aging networks suggesting a "weak link theory of aging". This theory suggests that age-related random damage primarily affects the overwhelming majority of the low affinity, transient interactions (weak links) in cellular networks leading to increased noise, destabilization and diversity. These processes may be further amplified by age-specific network remodelling and by the sequestration of weakly linked cellular proteins to protein aggregates of aging cells. Chaperones are weakly linked hubs (i.e., network elements with a large number of connections) and inter-modular bridge elements of protein-protein interaction, signalling and mitochondrial networks. As aging proceeds, the increased overload of damaged proteins is an especially important element contributing to cellular disintegration and destabilization. Additionally, chaperone overload may contribute to the increase of "noise" in aging cells, which leads to an increased stochastic resonance resulting in a deficient discrimination between signals and noise. Chaperone- and other multi-target therapies, which restore the missing weak links in aging cellular networks, may emerge as important anti-aging interventions. PMID:16814508

  4. Chaperones in hepatitis C virus infection

    PubMed Central

    Khachatoorian, Ronik; French, Samuel W

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses. PMID:26783419

  5. Chaperones in control of protein disaggregation

    PubMed Central

    Liberek, Krzysztof; Lewandowska, Agnieszka; Ziętkiewicz, Szymon

    2008-01-01

    The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates. PMID:18216875

  6. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    PubMed Central

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  7. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

    PubMed

    Woodford, Mark R; Dunn, Diana M; Blanden, Adam R; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A; Loh, Stewart N; Bourboulia, Dimitra; Schmidt, Laura S; Marston Linehan, W; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  8. Allostery in the Hsp70 chaperone proteins

    PubMed Central

    Zuiderweg, Erik R.P.; Bertelsen, Eric B.; Rousaki, Aikaterini; Mayer, Matthias P.; Gestwicki, Jason E.; Ahmad, Atta

    2013-01-01

    Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed. PMID:22576356

  9. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei.

    PubMed

    Burger, Adélle; Ludewig, Michael H; Boshoff, Aileen

    2014-01-01

    The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c. PMID:24707395

  10. Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code

    PubMed Central

    Cloutier, Philippe; Coulombe, Benoit

    2015-01-01

    Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point toward the more ambitious endeavor of deciphering the “chaperone code”. PMID:23459247

  11. Degradation of AF1Q by chaperone-mediated autophagy

    SciTech Connect

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru; Li, Huanjie; Cui, Taixing; Li Wang, Xing; Tang, Dongqi; Ji, Chunyan

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  12. The essential functions of endoplasmic reticulum chaperones in hepatic lipid metabolism.

    PubMed

    Zhang, LiChun; Wang, Hong-Hui

    2016-07-01

    The endoplasmic reticulum (ER) is an essential organelle for protein and lipid synthesis in hepatocytes. ER homeostasis is vital to maintain normal hepatocyte physiology. Perturbed ER functions causes ER stress associated with accumulation of unfolded protein in the ER that activates a series of adaptive signalling pathways, termed unfolded protein response (UPR). The UPR regulates ER chaperone levels to preserve ER protein-folding environment to protect the cell from ER stress. Recent findings reveal an array of ER chaperones that alter the protein-folding environment in the ER of hepatocytes and contribute to dysregulation of hepatocyte lipid metabolism and liver disease. In this review, we will discuss the specific functions of these chaperones in regulation of lipid metabolism, especially de novo lipogenesis and lipid transport and demonstrate their homeostatic role not only for ER-protein synthesis but also for lipid metabolism in hepatocyte. PMID:27133206

  13. CHIP: a co-chaperone for degradation by the proteasome.

    PubMed

    Edkins, Adrienne L

    2015-01-01

    Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfil well-defined roles in protein folding and conformational stability via ATP dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23 and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone mediated folding process. However, chaperones are also involved in ubiquitin-mediated proteasomal degradation of client proteins. Similar to folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C terminal Hsp70 binding protein (CHIP). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome system. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation. PMID:25487024

  14. Emerging novel concept of chaperone therapies for protein misfolding diseases

    PubMed Central

    SUZUKI, Yoshiyuki

    2014-01-01

    Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases. PMID:24814990

  15. β-asarone increases MEF2D and TH levels and reduces α-synuclein level in 6-OHDA-induced rats via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway: Chaperone-mediated autophagy activation, macroautophagy inhibition and HSP70 up-expression.

    PubMed

    Huang, Liping; Deng, Minzhen; He, Yuping; Lu, Shiyao; Liu, Shu; Fang, Yongqi

    2016-10-15

    Inactive myocyte enhancer factor 2D (MEF2D) and alpha-synuclein (α-syn) aggregation will cause neuronal death. MEF2D or α-syn degradation is also associated with macroautophagy, chaperone-mediated autophagy (CMA) and heat-shock protein 70 (HSP70). We found that β-asarone had positive effects on treating 6-hydroxydopamine (6-OHDA)-induced rats, but mechanisms of β-asarone affecting on MEF2D and α-syn via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway remain unclear. Unilateral 6-OHDA injection into the medial forebrain bundle was used to create PD rats, which were divided into four groups and administered for 30days: 6-OHDA model group, MEF2D inhibitor-treated group (SB203580, 0.5mg/kg, i.p.), MEF2D activator-treated group (LiCl, 100mg/kg, i.p.), β-asarone-treated group (15mg/kg, p.o.). Expressions of tyrosine hydroxylase (TH), α-syn, heat-shock cognate protein 70 (HSC70), lysosome-associated membrane protein type 2a (LAMP-2A), MEF2D, HSP70, Beclin-1, light chain 3B (LC3B) and p62 in the mesencephalon were measured after 30-day administration. α-syn, Beclin-1 and LC3B levels were higher in the 6-OHDA model group, while TH, MEF2D, HSC70, LAMP-2A, p62 levels were lower compared to the sham-operated group. Our results also showed thatβ-asarone treatment reduced protein and mRNA levels of α-syn, Beclin-1 and LC3B, but increased HSP70, TH, MEF2D, HSC70, LAMP-2A and p62 levels compared to the 6-OHDA model group. Additionally, certain correlations among α-syn, TH, Beclin-1, LC3B, p62, HSP70, LAMP-2A and MEF2D were also discovered in this study. These findings suggested that β-asarone treatment could increase MEF2D and TH as well as reduce α-syn to protect against 6-OHDA induced damage in PD rat mesencephalon via modulating the HSP70/MAPK/MEF2D/Beclin-1 pathway. PMID:27444243

  16. Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli.

    PubMed

    Sonoda, Hiroyuki; Kumada, Yoichi; Katsuda, Tomohisa; Yamaji, Hideki

    2011-04-01

    The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective. PMID:21324738

  17. Calcium binding chaperones of the endoplasmic reticulum.

    PubMed

    Coe, Helen; Michalak, Marek

    2009-01-01

    The endoplasmic reticulum is a major Ca(2+) store of the cell that impacts many cellular processes within the cell. The endoplasmic reticulum has roles in lipid and sterol synthesis, protein folding, post-translational modification and secretion and these functions are affected by intraluminal endoplasmic reticulum Ca(2+). In the endoplasmic reticulum there are several Ca(2+) buffering chaperones including calreticulin, Grp94, BiP and protein disulfide isomerase. Calreticulin is one of the major Ca(2+) binding/buffering chaperones in the endoplasmic reticulum. It has a critical role in Ca(2+) signalling in the endoplasmic reticulum lumen and this has significant impacts on many Ca(2+)-dependent pathways including control of transcription during embryonic development. In addition to Ca(2+) buffering, calreticulin plays important role in the correct folding and quality control of newly synthesized glycoproteins. PMID:20093733

  18. The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

    PubMed Central

    Lu, Xin-an; Wang, Xiaofeng; Zhuo, Wei; Jia, Lin; Jiang, Yushan; Fu, Yan; Luo, Yongzhang

    2013-01-01

    It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr115/Thr425/Thr603, of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a ‘phosphorylation switch’ for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells. PMID:24117238

  19. Tau triage decisions mediated by the chaperone network.

    PubMed

    Cook, Casey; Petrucelli, Leonard

    2013-01-01

    The pathological accumulation of the microtubule-binding protein tau is linked to an increasing number of neurodegenerative conditions associated with aging, though the mechanisms by which tau accumulates in disease are unclear. In this review, we will summarize our previous research assessing the mechanism of action, as well as the therapeutic potential of Hsp90 inhibition for the treatment of tauopathies. Specifically, we describe the development of a high-throughput screening approach to identify and rank compounds, and demonstrate the selective elimination of aberrant p-tau species in the brain following treatment with an Hsp90 inhibitor. Additionally, we identify CHIP as an essential component of the Hsp90 chaperone complex that mediates tau degradation, and present evidence to suggest that CHIP functions to identify and sequester neurotoxic tau species. Finally, we discuss recent data identifying an additional mechanism by which CHIP modulates protein triage decisions involving Hsp90. Specifically, CHIP indirectly regulates Hsp90 chaperone activity by modulating steady-state levels of the Hsp90 deacetylase, HDAC6, thus influencing both the acetylation state and function of Hsp90. Thus future research directions will focus on the manipulation of this network to promote degradation of pathogenic tau species in disease. PMID:22596270

  20. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production.

    PubMed

    Martínez-Alonso, Mónica; Villaverde, Antonio; Ferrer-Miralles, Neus

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  1. Suppression of protein aggregation by chaperone modification of high molecular weight complexes

    PubMed Central

    Labbadia, John; Novoselov, Sergey S.; Bett, John S.; Weiss, Andreas; Paganetti, Paolo; Bates, Gillian P.

    2012-01-01

    Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington’s disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington’s disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington’s disease. PMID:22396390

  2. Endoplasmic reticulum chaperones and oxidoreductases: critical regulators of tumor cell survival and immunorecognition.

    PubMed

    Gutiérrez, Tomás; Simmen, Thomas

    2014-01-01

    Endoplasmic reticulum (ER) chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed "bulk flow," ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER by their substrates. Solid tumors are characterized by the increased production of reactive oxygen species (ROS), combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response upregulate their target genes. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the folding of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an "eat-me" signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI), Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies. PMID:25386408

  3. Suppression of protein aggregation by chaperone modification of high molecular weight complexes.

    PubMed

    Labbadia, John; Novoselov, Sergey S; Bett, John S; Weiss, Andreas; Paganetti, Paolo; Bates, Gillian P; Cheetham, Michael E

    2012-04-01

    Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease. PMID:22396390

  4. Regulation of organismal proteostasis by trans-cellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Porter, Robert S.; Morimoto, Richard I.

    2013-01-01

    Summary A major challenge for metazoans is to ensure that different tissues each expressing distinctive proteomes are, nevertheless, well protected at an organismal level from proteotoxic stress. We have examined this and show that expression of endogenous metastable protein sensors in muscle cells induces a systemic stress response throughout multiple tissues of C. elegans. Suppression of misfolding in muscle cells can be achieved not only by enhanced expression of HSP90 in muscle cells, but as effective by elevated expression of HSP90 in intestine or neuronal cells. This cell-non-autonomous control of HSP90 expression relies upon transcriptional feedback between somatic tissues that is regulated by the FoxA transcription factor PHA-4. This trans-cellular chaperone signaling response maintains organismal proteostasis when challenged by a local tissue imbalance in folding and provides the basis for a novel form of organismal stress sensing surveillance. PMID:23746847

  5. A photoconvertible fluorescent reporter to track chaperone-mediated autophagy

    PubMed Central

    Koga, Hiroshi; Martinez-Vicente, Marta; Macian, Fernando; Verkhusha, Vladislav V; Cuervo, Ana Maria

    2012-01-01

    Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble proteins in lysosomes. CMA contributes to cellular quality control and is activated as part of the cellular response to different stressors. Defective CMA has been identified in aging and different age-related diseases. Until now, CMA activity could only be measured in vitro upon isolation of lysosomes. Here we report the development of a photoconvertible fluorescent reporter that allows monitoring of CMA activity in living cells. Activation of CMA increases the association of the reporter with lysosomes which are visualized as a change in the intracellular fluorescence. The CMA reporter can be utilized in a broad variety of cells and is suitable for high-content microscopy. Using this reporter, we find that levels of basal and inducible CMA activity are cell-type dependent and we have identified an upregulation of this pathway in response to the catalytic inhibition of the proteasome. PMID:21750540

  6. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

    PubMed

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  7. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  8. Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models.

    PubMed

    Sanchez-Martinez, Alvaro; Beavan, Michelle; Gegg, Matthew E; Chau, Kai-Yin; Whitworth, Alexander J; Schapira, Anthony H V

    2016-01-01

    GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD. PMID:27539639

  9. Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models

    PubMed Central

    Sanchez-Martinez, Alvaro; Beavan, Michelle; Gegg, Matthew E.; Chau, Kai-Yin; Whitworth, Alexander J.; Schapira, Anthony H. V.

    2016-01-01

    GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD. PMID:27539639

  10. Capturing the misfolds : chaperone-peptide-binding motifs.

    SciTech Connect

    Joachimiak, A.; Center for Mechanistic Biology and Biotechnology

    1997-06-01

    Recently, the crystal structure of the N-terminal fragment of human Hsp90-alpha chaperone and its complex with geldanamycin and the crystal structure of the N-terminal domain of yeast Hsp90 have been determined at high resolution. These structures reveal features that shed new light on the Hsp90 chaperone-protein interactions.

  11. Mitochondrial chaperones may be targets for anti-cancer drugs

    Cancer.gov

    Scientists at NCI have found that a mitochondrial chaperone protein, TRAP1, may act indirectly as a tumor suppressor as well as a novel target for developing anti-cancer drugs. Chaperone proteins, such as TRAP1, help other proteins adapt to stress, but sc

  12. Modulation of human IAPP fibrillation: cosolutes, crowders and chaperones.

    PubMed

    Gao, Mimi; Estel, Kathrin; Seeliger, Janine; Friedrich, Ralf P; Dogan, Susanne; Wanker, Erich E; Winter, Roland; Ebbinghaus, Simon

    2015-04-01

    The cellular environment determines the structure and function of proteins. Marginal changes of the environment can severely affect the energy landscape of protein folding. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). By applying biophysical methods such as thioflavin T fluorescence spectroscopy, fluorescence anisotropy, total reflection Fourier-transform infrared spectroscopy, circular dichroism spectroscopy and atomic force microscopy we analyse and compare their inhibition mechanism. We demonstrate that the fibrillation reaction of human IAPP is strongly inhibited by formation of globular, amorphous assemblies by both, the pharmacological and the protein chaperones. We studied the inhibition mechanism under cell-like conditions by using the artificial crowding agents Ficoll 70 and sucrose. Under such conditions the suppressive effect of proline was decreased, whereas the pharmacological chaperone remains active. PMID:25406896

  13. Toward Instituting a Chaperone Policy in Outpatient Pediatric Clinics

    ERIC Educational Resources Information Center

    Feldman, Kenneth W.; Jenkins, Carol; Laney, Tyler; Seidel, Kristy

    2009-01-01

    Objectives: We sought to evaluate child, parent and medical provider preferences for chaperones for outpatient encounters and to evaluate the acceptability and frequency of utilization following institution of a chaperone policy. Secondarily, we sought to understand what medical history and examinations teens consider "sensitive." Design: We…

  14. Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum

    PubMed Central

    Teixeira, Filipa; Castro, Helena; Cruz, Tânia; Tse, Eric; Koldewey, Philipp; Southworth, Daniel R.; Tomás, Ana M.; Jakob, Ursula

    2015-01-01

    Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx’s ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx’s chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection. PMID:25646478

  15. Myopathy-causing mutations in an HSP40 chaperone disrupt processing of specific client conformers.

    PubMed

    Stein, Kevin C; Bengoechea, Rocio; Harms, Matthew B; Weihl, Conrad C; True, Heather L

    2014-07-25

    The molecular chaperone network protects against the toxic misfolding and aggregation of proteins. Disruption of this network leads to a variety of protein conformational disorders. One such example recently discovered is limb-girdle muscular dystrophy type 1D (LGMD1D), which is caused by mutation of the HSP40 chaperone DNAJB6. All LGMD1D-associated mutations localize to the conserved G/F domain of DNAJB6, but the function of this domain is largely unknown. Here, we exploit the yeast HSP40 Sis1, which has known aggregation-prone client proteins, to gain insight into the role of the G/F domain and its significance in LGMD1D pathogenesis. Strikingly, we demonstrate that LGMD1D mutations in a Sis1-DNAJB6 chimera differentially impair the processing of specific conformers of two yeast prions, [RNQ+] and [PSI+]. Importantly, these differences do not simply correlate to the sensitivity of these prion strains to changes in chaperone levels. Additionally, we analyzed the effect of LGMD1D-associated DNAJB6 mutations on TDP-43, a protein known to form inclusions in LGMD1D. We show that the DNAJB6 G/F domain mutants disrupt the processing of nuclear TDP-43 stress granules in mammalian cells. These data suggest that the G/F domain mediates chaperone-substrate interactions in a manner that extends beyond recognition of a particular client and to a subset of client conformers. We propose that such selective chaperone disruption may lead to the accumulation of toxic aggregate conformers and result in the development of LGMD1D and perhaps other protein conformational disorders. PMID:24920671

  16. Interplay between chaperones and protein disorder promotes the evolution of protein networks.

    PubMed

    Pechmann, Sebastian; Frydman, Judith

    2014-06-01

    Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular

  17. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks

    PubMed Central

    Pechmann, Sebastian; Frydman, Judith

    2014-01-01

    Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular

  18. CSPα—chaperoning presynaptic proteins

    PubMed Central

    Donnelier, Julien; Braun, Janice E. A.

    2014-01-01

    Synaptic transmission relies on precisely regulated and exceedingly fast protein-protein interactions that involve voltage-gated channels, the exocytosis/endocytosis machinery as well as signaling pathways. Although we have gained an ever more detailed picture of synaptic architecture much remains to be learned about how synapses are maintained. Synaptic chaperones are “folding catalysts” that preserve proteostasis by regulating protein conformation (and therefore protein function) and prevent unwanted protein-protein interactions. Failure to maintain synapses is an early hallmark of several degenerative diseases. Cysteine string protein (CSPα) is a presynaptic vesicle protein and molecular chaperone that has a central role in preventing synaptic loss and neurodegeneration. Over the past few years, a number of different “client proteins” have been implicated as CSPα substrates including voltage-dependent ion channels, signaling proteins and proteins critical to the synaptic vesicle cycle. Here we review the ion channels and synaptic protein complexes under the influence of CSPα and discuss gaps in our current knowledge. PMID:24808827

  19. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  20. THE PROTEIN TARGETING FACTOR GET3 FUNCTIONS AS AN ATP-INDEPENDENT CHAPERONE UNDER OXIDATIVE STRESS CONDITIONS

    PubMed Central

    Voth, Wilhelm; Schick, Markus; Gates, Stephanie; Li, Sheng; Vilardi, Fabio; Gostimskaya, Irina; Southworth, Daniel R.; Schwappach, Blanche; Jakob, Ursula

    2014-01-01

    Summary Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP-levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under non-stress conditions, turns into an effective ATP-in dependent chaperone when oxidized. Activation of Get3’s chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage. PMID:25242142

  1. Expression and variability of molecular chaperones in the sugarcane expressome.

    PubMed

    Borges, Júlio C; Cagliari, Thiago C; Ramos, Carlos H I

    2007-04-01

    Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. PMID:16687190

  2. Apg-2 has a chaperone-like activity similar to Hsp110 and is overexpressed in hepatocellular carcinomas.

    PubMed

    Gotoh, Kazuhisa; Nonoguchi, Kohsuke; Higashitsuji, Hiroaki; Kaneko, Yoshiyuki; Sakurai, Toshiharu; Sumitomo, Yasuhiko; Itoh, Katsuhiko; Subjeck, John R; Fujita, Jun

    2004-02-27

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities. PMID:14987991

  3. Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

    PubMed Central

    Sichting, Martin; Mokranjac, Dejana; Azem, Abdussalam; Neupert, Walter; Hell, Kai

    2005-01-01

    Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones. PMID:15719019

  4. Signal peptide protection by specific chaperone

    SciTech Connect

    Genest, Olivier; Seduk, Farida; Ilbert, Marianne; Mejean, Vincent; Iobbi-Nivol, Chantal . E-mail: iobbi@ibsm.cnrs-mrs.fr

    2006-01-20

    TorD is the private chaperone of TorA, a periplasmic respiratory molybdoenzyme of Escherichia coli. In this study, it is demonstrated that TorD is required to maintain the integrity of the twin-arginine signal sequence of the cytoplasmic TorA precursors. In the absence of TorD, 35 out of the 39 amino acid residues of the signal peptide were lost and the proteolysis of the N-terminal extremity of TorA precursors was not prevented by the molybdenum cofactor insertion. We thus propose that one of the main roles of TorD is to protect the TorA signal peptide to allow translocation of the enzyme by the TAT system.

  5. Stress chaperone mortalin regulates human melanogenesis.

    PubMed

    Wadhwa, Renu; Priyandoko, Didik; Gao, Ran; Widodo, Nashi; Nigam, Nupur; Li, Ling; Ahn, Hyo Min; Yun, Chae-Ok; Ando, Nobuhiro; Mahe, Christian; Kaul, Sunil C

    2016-07-01

    In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics. PMID:27056733

  6. Promiscuous Substrate Recognition in Folding and Assembly Activities of the Trigger Factor Chaperone

    SciTech Connect

    Martinez-Hackert, E.; Hendrickson, W

    2009-01-01

    Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.

  7. The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding.

    PubMed

    Baaklini, Imad; Wong, Michael J H; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C

    2012-12-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  8. The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding*

    PubMed Central

    Baaklini, Imad; Wong, Michael J. H.; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C.

    2012-01-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  9. Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders

    PubMed Central

    Parenti, Giancarlo; Andria, Generoso; Valenzano, Kenneth J

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches. PMID:25881001

  10. InvB is a type III secretion chaperone specific for SspA.

    PubMed

    Bronstein, P A; Miao, E A; Miller, S I

    2000-12-01

    A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium. PMID:11073906

  11. Molecular chaperone-mediated nuclear protein dynamics.

    PubMed

    Echtenkamp, Frank J; Freeman, Brian C

    2014-05-01

    Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus. PMID:24694369

  12. Stratified analysis of lectin-like chaperones in the folding disease-related metabolic syndrome rat model.

    PubMed

    Hirano, Makoto; Imagawa, Ayami; Totani, Kiichiro

    2016-09-01

    The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels. PMID:27425249

  13. The archaeal molecular chaperone machine: peculiarities and paradoxes.

    PubMed Central

    Macario, A J; de Macario, E C

    1999-01-01

    A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains. PMID:10430558

  14. Histone Chaperone HIRA in Regulation of Transcription Factor RUNX1.

    PubMed

    Majumder, Aditi; Syed, Khaja Mohieddin; Joseph, Sunu; Scambler, Peter J; Dutta, Debasree

    2015-05-22

    RUNX1 (Runt-related transcription factor 1) is indispensable for the generation of hemogenic endothelium. However, the regulation of RUNX1 during this developmental process is poorly understood. We investigated the role of the histone chaperone HIRA (histone cell cycle regulation-defective homolog A) from this perspective and report that HIRA significantly contributes toward the regulation of RUNX1 in the transition of differentiating mouse embryonic stem cells from hemogenic to hematopoietic stage. Direct interaction of HIRA and RUNX1 activates the downstream targets of RUNX1 implicated in generation of hematopoietic stem cells. At the molecular level, HIRA-mediated incorporation of histone H3.3 variant within the Runx1 +24 mouse conserved noncoding element is essential for the expression of Runx1 during endothelial to hematopoietic transition. An inactive chromatin at the intronic enhancer of Runx1 in absence of HIRA significantly repressed the transition of cells from hemogenic to hematopoietic fate. We expect that the HIRA-RUNX1 axis might open up a novel approach in understanding leukemogenesis in future. PMID:25847244

  15. The histone chaperone CAF-1 safeguards somatic cell identity.

    PubMed

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Euong Ang, Cheen; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter J; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2015-12-10

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  16. The histone chaperone CAF-1 safeguards somatic cell identity

    PubMed Central

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y.; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2016-01-01

    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  17. Bacterial Discrimination by FISH using Molecular Chaperon GroE

    NASA Astrophysics Data System (ADS)

    Nakamura, T.; Maruyama, A.; Kurusu, Y.

    2004-12-01

    FISH(Fluorescence In Situ hybridization) is a powerful method for the analysis of the phylogenetic classification of microorganism in the environment. In many cases, 16s rRNA sequences of microorganisms are employed as target probe. Here we showed that novel probe was used in FISH in order to discriminate among the bacteria including psychrophile, mesophile, and thermophile. Molecular Chaperon GroE is a best characterized protein based on Escherichia coli and essential for bacterial proliferation. In E. coli, the amount of GroEL protein per cell reaches to about 5% of total cellualr protein at heat-shock response. This response occurred at transcription levels, the amount of groEL mRNA increases at about 10-fold per cell, reaches to 0.4% of total synthesized RNA. Therefore, we considered that groEL gene was employed FISH analysis as a target probe. Moreover, we found that Gly-Gly-Met (GGM) repeats in the carboxy-terminal of GroEL strongly conserved among psychrophile and mesophile, but not thermophile. In this report, we attempted to discriminate among the bacteria including psychrophile, mesophile, and thermophile by FISH using the specific sequence of GroEL as a probe. Furthermore, we proposed the novel phylogenetic trees based on the amino acids sequences of carboxy-terminal of GroEL for bacterial evolution by temperature adaptation.

  18. Inhibitors of the AAA+ Chaperone p97

    PubMed Central

    Chapman, Eli; Maksim, Nick; de la Cruz, Fabian; La Clair, James J.

    2015-01-01

    It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA) more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®), which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+) chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and physiology. PMID

  19. Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol.

    PubMed

    Ayala, Pedro; Montenegro, José; Vivar, Raúl; Letelier, Alan; Urroz, Pablo Aránguiz; Copaja, Miguel; Pivet, Deisy; Humeres, Claudio; Troncoso, Rodrigo; Vicencio, José Miguel; Lavandero, Sergio; Díaz-Araya, Guillermo

    2012-02-01

    Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In the human heart, ischemia/reperfusion has been correlated to ER stress, and several markers of the unfolded protein response (UPR) participate during cardiac remodeling and fibrosis. Here, we used isoproterenol (ISO) injection as a model for in vivo cardiac fibrosis. ISO induced significant cardiomyocyte loss and collagen deposition in the damaged areas of the endocardium. These responses were accompanied by an increase in the protein levels of the luminal ER chaperones BIP and PDI, as well as an increase in the UPR effector CHOP. The use of the chemical chaperone 4-phenylbutyric acid (4-PBA) prevented the activation of the UPR, the increase in luminal chaperones and also, leads to decreased collagen deposition, cardiomyocyte loss into the damaged zones. Our results suggest that cardiac damage and fibrosis induced in vivo by the beta-adrenergic agonist ISO are tightly related to ER stress signaling pathways, and that increasing the ER luminal folding capacity with exogenously administrated 4-PBA is a powerful strategy for preventing the development of cardiac fibrosis. Additionally, 4-PBA might prevent the loss of cardiomyocytes. Our data suggests that the attenuation of ER stress pathways with pharmacological compounds such as the chemical chaperone 4-PBA can prevent the development of cardiac fibrosis and adverse remodeling. PMID:22101259

  20. A Novel Method for Assessing the Chaperone Activity of Proteins.

    PubMed

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families-molecules expressed during adverse conditions, infection, and diseases-chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  1. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  2. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates.

    PubMed

    Braselmann, Esther; Chaney, Julie L; Champion, Matthew M; Clark, Patricia L

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress. PMID:27626276

  3. Chaperone-assisted selective autophagy is essential for muscle maintenance.

    PubMed

    Arndt, Verena; Dick, Nikolaus; Tawo, Riga; Dreiseidler, Michael; Wenzel, Daniela; Hesse, Michael; Fürst, Dieter O; Saftig, Paul; Saint, Robert; Fleischmann, Bernd K; Hoch, Michael; Höhfeld, Jörg

    2010-01-26

    How are biological structures maintained in a cellular environment that constantly threatens protein integrity? Here we elucidate proteostasis mechanisms affecting the Z disk, a protein assembly essential for actin anchoring in striated muscles, which is subjected to mechanical, thermal, and oxidative stress during contraction [1]. Based on the characterization of the Drosophila melanogaster cochaperone Starvin (Stv), we define a conserved chaperone machinery required for Z disk maintenance. Instead of keeping Z disk proteins in a folded conformation, this machinery facilitates the degradation of damaged components, such as filamin, through chaperone-assisted selective autophagy (CASA). Stv and its mammalian ortholog BAG-3 coordinate the activity of Hsc70 and the small heat shock protein HspB8 during disposal that is initiated by the chaperone-associated ubiquitin ligase CHIP and the autophagic ubiquitin adaptor p62. CASA is thus distinct from chaperone-mediated autophagy, previously shown to facilitate the ubiquitin-independent, direct translocation of a client across the lysosomal membrane [2]. Impaired CASA results in Z disk disintegration and progressive muscle weakness in flies, mice, and men. Our findings reveal the importance of chaperone-assisted degradation for the preservation of cellular structures and identify muscle as a tissue that highly relies on an intact proteostasis network, thereby shedding light on diverse myopathies and aging. PMID:20060297

  4. Plasmodium falciparum-encoded exported hsp70/hsp40 chaperone/co-chaperone complexes within the host erythrocyte.

    PubMed

    Külzer, Simone; Charnaud, Sarah; Dagan, Tal; Riedel, Jan; Mandal, Pradipta; Pesce, Eva R; Blatch, Gregory L; Crabb, Brendan S; Gilson, Paul R; Przyborski, Jude M

    2012-11-01

    Malaria parasites modify their host cell, the mature human erythrocyte. We are interested in the molecules mediating these processes, and have recently described a family of parasite-encoded heat shock proteins (PfHsp40s) that are targeted to the host cell, and implicated in host cell modification. Hsp40s generally function as co-chaperones of members of the Hsp70 family, and until now it was thought that human Hsp70 acts as the PfHsp40 interaction partner within the host cell. Here we revise this hypothesis, and identify and characterize an exported parasite-encoded Hsp70, referred to as PfHsp70-x. PfHsp70-x is exported to the host erythrocyte where it forms a complex with PfHsp40s in structures known as J-dots, and is closely associated with PfEMP1. Interestingly, Hsp70-x is encoded only by parasite species that export the major virulence factor EMP1, implying a possible role for Hsp70-x in EMP1 presentation at the surface of the infected erythrocyte. Our data strongly support the presence of parasite-encoded chaperone/co-chaperone complexes within the host erythrocyte, which are involved in protein traffic through the host cell. The host-pathogen interaction within the infected erythrocyte is more complex than previously thought, and is driven notonly by parasite co-chaperones, but also by the parasite-encoded chaperone Hsp70-x itself. PMID:22925632

  5. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    PubMed

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone. PMID:26619265

  6. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    PubMed

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. PMID:26991466

  7. Pathways of allosteric regulation in Hsp70 chaperones.

    PubMed

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s. PMID:26383706

  8. Specific chaperones and regulatory domains in control of amyloid formation.

    PubMed

    Landreh, Michael; Rising, Anna; Presto, Jenny; Jörnvall, Hans; Johansson, Jan

    2015-10-30

    Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology. PMID:26354437

  9. A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies.

    PubMed

    Luan, Zhuo; Higaki, Katsumi; Aguilar-Moncayo, Matilde; Li, Linjing; Ninomiya, Haruaki; Nanba, Eiji; Ohno, Kousaku; García-Moreno, M Isabel; Ortiz Mellet, Carmen; García Fernández, José M; Suzuki, Yoshiyuki

    2010-11-22

    Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms. PMID:21064079

  10. Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis.

    PubMed

    Leng, Xiangpeng; Mu, Qian; Wang, Xiaomin; Li, Xiaopeng; Zhu, Xudong; Shangguan, Lingfei; Fang, Jinggui

    2015-11-01

    With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu(2+) were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied. PMID:26054906

  11. Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane

    PubMed Central

    Han, Gayoung A.; Malintan, Nancy T.; Saw, Ner Mu Nar; Li, Lijun; Han, Liping; Meunier, Frederic A.; Collins, Brett M.; Sugita, Shuzo

    2011-01-01

    Munc18-1 plays pleiotropic roles in neurosecretion by acting as 1) a molecular chaperone of syntaxin-1, 2) a mediator of dense-core vesicle docking, and 3) a priming factor for soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion. However, how these functions are executed and whether they are correlated remains unclear. Here we analyzed the role of the domain-1 cleft of Munc18-1 by measuring the abilities of various mutants (D34N, D34N/M38V, K46E, E59K, K46E/E59K, K63E, and E66A) to bind and chaperone syntaxin-1 and to restore the docking and secretion of dense-core vesicles in Munc18-1/-2 double-knockdown cells. We identified striking correlations between the abilities of these mutants to bind and chaperone syntaxin-1 with their ability to restore vesicle docking and secretion. These results suggest that the domain-1 cleft of Munc18-1 is essential for binding to syntaxin-1 and thereby critical for its chaperoning, docking, and secretory functions. Our results demonstrate that the effect of the alleged priming mutants (E59K, D34N/M38V) on exocytosis can largely be explained by their reduced syntaxin-1–chaperoning functions. Finally, our data suggest that the intracellular expression and distribution of syntaxin-1 determines the level of dense-core vesicle docking. PMID:21900502

  12. Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

    PubMed

    Kusek, Justyna; Yang, Qiong; Witek, Martin; Gruber, Christian W; Nanoff, Christian; Freissmuth, Michael

    2015-01-01

    Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress. PMID:25354767

  13. Pharmacological chaperones for human α-N-acetylgalactosaminidase.

    PubMed

    Clark, Nathaniel E; Metcalf, Matthew C; Best, Daniel; Fleet, George W J; Garman, Scott C

    2012-10-23

    Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases. PMID:23045655

  14. Pharmacological chaperones for human α-N-acetylgalactosaminidase

    PubMed Central

    Clark, Nathaniel E.; Metcalf, Matthew C.; Best, Daniel; Fleet, George W. J.; Garman, Scott C.

    2012-01-01

    Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases. PMID:23045655

  15. Super Spy variants implicate flexibility in chaperone action

    PubMed Central

    Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

    2014-01-01

    Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

  16. Chaperone-assisted translocation of flexible polymers in three dimensions

    NASA Astrophysics Data System (ADS)

    Suhonen, P. M.; Linna, R. P.

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single site or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain β ≈1.26 for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be explained by the additional friction due to binding particles. The multiple-site binding leads to translocation the dynamics of which is mainly determined by the trans side. For this process we obtain β ≈1.36 . This value can be explained by our derivation of β =4 /3 for constant-bias translocation, where translocated polymer segments form a globule on the trans side. Our results pave the way for understanding and utilizing chaperone-assisted translocation where variations in microscopic details lead to rich variations in the emerging dynamics.

  17. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  18. Hsp100/ClpB Chaperone Function and Mechanism

    SciTech Connect

    Vierling, Elizabeth

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  19. Hsp70-Hsp40 Chaperone Complex Functions in Controlling Polarized Growth by Repressing Hsf1-Driven Heat Stress-Associated Transcription

    PubMed Central

    Liu, Jianhua; Oliferenko, Snezhana

    2013-01-01

    How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments. PMID:24146635

  20. The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression.

    PubMed

    Bolt, G

    2001-01-01

    The present study examines the coprecipitation of measles virus (MV) glycoproteins with host cell endoplasmic reticulum (ER) chaperone proteins. Both the haemagglutinin (H) and fusion (F) glycoproteins interacted with calnexin and GRP78, whereas interaction with calreticulin was only demonstrated for the H glycoprotein. The alpha-glucosidase inhibitor castanospermine reduced and delayed the association of F proteins with calnexin. We have previously shown that alpha-glucosidase activity is important for the functionality and antigenicity of the MV F glycoprotein and for release of MV particles from infected cells. Thus, interaction with calnexin appears vital for processing of nascent MV F protein into its functional conformation. In contrast to many other viral glycoproteins, a substantial proportion of the pulsed MV glycoproteins remained associated with ER chaperones for more than 2(1/2) h. Thus, the slow and incomplete migration of MV glycoproteins to the cell surface may result from their retention by ER chaperones, probably due to malfolding. MV infection upregulated the cellular expression of calreticulin and GRP78 and also increased their presence at the cell surface. The chaperone proteins are involved in a wide range of cellular processes, and their induction by MV may play a role for the pathogenesis of measles and its sequelae. PMID:11765911

  1. Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

    PubMed Central

    Asim, Mohammad; Massie, Charles E.; Orafidiya, Folake; Pértega-Gomes, Nelma; Warren, Anne Y.; Esmaeili, Mohsen; Selth, Luke A.; Zecchini, Heather I.; Luko, Katarina; Qureshi, Arham; Baridi, Ajoeb; Menon, Suraj; Madhu, Basetti; Escriu, Carlos; Lyons, Scott; Vowler, Sarah L.; Zecchini, Vincent R.; Shaw, Greg; Hessenkemper, Wiebke; Russell, Roslin; Mohammed, Hisham; Stefanos, Niki; Lynch, Andy G.; Grigorenko, Elena; D’Santos, Clive; Taylor, Chris; Lamb, Alastair; Sriranjan, Rouchelle; Yang, Jiali; Stark, Rory; Dehm, Scott M.; Rennie, Paul S.; Carroll, Jason S.; Griffiths, John R.; Tavaré, Simon; Mills, Ian G.; McEwan, Iain J.; Baniahmad, Aria; Tilley, Wayne D.; Neal, David E.

    2016-01-01

    Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. PMID:26657335

  2. Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type.

    PubMed

    Jorge-Finnigan, Ana; Brasil, Sandra; Underhaug, Jarl; Ruíz-Sala, Pedro; Merinero, Begoña; Banerjee, Ruma; Desviat, Lourdes R; Ugarte, Magdalena; Martinez, Aurora; Pérez, Belén

    2013-09-15

    Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations. PMID:23674520

  3. Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type

    PubMed Central

    Jorge-Finnigan, Ana; Brasil, Sandra; Underhaug, Jarl; Ruíz-Sala, Pedro; Merinero, Begoña; Banerjee, Ruma; Desviat, Lourdes R.; Ugarte, Magdalena; Martinez, Aurora; Pérez, Belén

    2013-01-01

    Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations. PMID:23674520

  4. The Hsp70 and Hsp40 Chaperones Influence Microtubule Stability in Chlamydomonas

    PubMed Central

    Silflow, Carolyn D.; Sun, Xiaoqing; Haas, Nancy A.; Foley, Joseph W.; Lefebvre, Paul A.

    2011-01-01

    Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70–Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex. PMID:21940683

  5. Coffee enhances the expression of chaperones and antioxidant proteins in rats with nonalcoholic fatty liver disease.

    PubMed

    Salomone, Federico; Li Volti, Giovanni; Vitaglione, Paola; Morisco, Filomena; Fogliano, Vincenzo; Zappalà, Agata; Palmigiano, Angelo; Garozzo, Domenico; Caporaso, Nicola; D'Argenio, Giuseppe; Galvano, Fabio

    2014-06-01

    Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee's beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2'-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver. PMID:24365744

  6. The Co-chaperone BAG2 Sweeps PHF Insoluble Tau from the Microtubule

    PubMed Central

    Carrettiero, Daniel C.; Hernandez, Israel; Neveu, Pierre; Papagiannakopoulos, Thales; Kosik, Kenneth S.

    2009-01-01

    Tau inclusions are a prominent feature of many neurodegenerative diseases including Alzheimer’s disease. Their accumulation in neurons as ubiquitinated filaments suggests a failure in the degradation limb of the Tau pathway. The components of a Tau protein triage system consisting of CHIP/Hsp70 and other chaperones have begun to emerge. However, the site of triage and the master regulatory elements are unknown. Here we report an elegant mechanism of Tau degradation involving the co-chaperone BAG2. The BAG2/Hsp70 complex is tethered to the microtubule and this complex can capture and deliver Tau to the proteasome for ubiquitin-independent degradation. This complex preferentially degrades sarkosyl insoluble Tau and phosphorylated Tau. BAG2 levels in cells are under the physiological control of the microRNA miR-128a, which can tune PHF Tau levels in neurons. Thus we propose that ubiquitinated Tau inclusions arise due to shunting of Tau degradation toward a less efficient ubiquitin-dependent pathway. PMID:19228967

  7. Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein*

    PubMed Central

    Stieren, Emily S.; El Ayadi, Amina; Xiao, Yao; Siller, Efraín; Landsverk, Megan L.; Oberhauser, Andres F.; Barral, José M.; Boehning, Darren

    2011-01-01

    Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis. PMID:21852239

  8. Effect of endogenous Hsp104 chaperone in yeast models of sporadic and familial Parkinson's disease.

    PubMed

    Gade, Vamshidhar R; Kardani, Jay; Roy, Ipsita

    2014-10-01

    Molecular chaperones constitute a major component of the cellular stress response machinery in neurodegenerative diseases, many of which are characterized by the misfolding and aggregation of endogenous cellular proteins into generic amyloid macrostructures. Heterologous expression of the yeast protein remodelling factor Hsp104 has been proposed as a possible therapeutic approach in such disease conditions. Hsp104 is unique in its ability to act as a protein 'disaggregase' by removing smaller units from amyloid fibrils and has no homologue in metazoa. The effect of Hsp104 is strongly modulated by its expression level. We show that at endogenous levels, the presence of Hsp104 has a deleterious effect on protein aggregation in two different strains of yeast. Overexpression of wild-type and mutant human α-synuclein in a well-validated yeast model of Parkinson's disease and in an isogenic Hsp104-deleted strain resulted in lower oxidative stress and reduced damage to cellular proteins in the latter case. This translated to lower cytotoxicity and increased cell viability. Endocytotic defect caused due to aggregation of α-syuclein was also rescued in cells lacking Hsp104. Our results show that the effect of overexpression of a chaperone on protein misfolding/aggregation cannot be predicted from its function in the host expression platform. PMID:25161148

  9. Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth

    PubMed Central

    Lv, Lei; Li, Dong; Zhao, Di; Lin, Ruiting; Chu, Yajing; Zhang, Heng; Zha, Zhengyu; Liu, Ying; Li, Zi; Xu, Yanping; Wang, Gang; Huang, Yiran; Xiong, Yue; Guan, Kun-Liang; Lei, Qun-Ying

    2016-01-01

    SUMMARY Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA. PMID:21700219

  10. Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures

    PubMed Central

    Bencomo, Alberto; Lara-Martínez, Reyna; Rivera-Marrero, Suchitil; Domínguez, Guadalupe; Pérez-Perera, Rafaela; Jiménez-García, Luis Felipe; Altamirano-Bustamante, Nelly F.; Diaz-Delgado, Massiel; Vedrenne, Fernand; Rivillas-Acevedo, Lina; Pasten-Hidalgo, Karina; Segura-Valdez, María de Lourdes; Islas-Andrade, Sergio; Garrido-Magaña, Eulalia; Perera-Pintado, Alejandro; Prats-Capote, Anaís; Rodríguez-Tanty, Chryslaine; Altamirano-Bustamante, Myriam M.

    2015-01-01

    The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are

  11. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  12. Large-scale gene expression profiling reveals physiological response to deletion of chaperone dnaKJ in Escherichia coli.

    PubMed

    Fan, Dongjie; Liu, Chuanpeng; Liu, Lushan; Zhu, Lingxiang; Peng, Fang; Zhou, Qiming

    2016-01-01

    Chaperone DnaK and its co-chaperone DnaJ plays various essential roles such as in assisting in the folding of nascent peptides, preventing protein aggregation and maintaining cellular protein homeostasis. Global transcriptional changes in vivo associated with deletion of dnaKJ were monitored using DNA microarray to elucidate the role of DnaKJ at the transcriptional level. Microarray profiling and bioinformatics analysis revealed that a few chaperone and protease genes, stress-related genes and genes involved in the tricarboxylic acid cycle and oxidative phosphorylation were up-regulated, whereas various transporter genes, pentose phosphate pathway and transcriptional regulation related genes were down-regulated. This study is the first to systematically analyze the alterations at the transcriptional level in vivo in deletion of dnaKJ. Fatty acid methyl esters analysis indicated that the amount of unsaturated fatty acid sharply increased and subcellular location prediction analysis showed a marked decrease in transcription of inner-membrane protein genes, which might have triggered the development of aberrant cell shape and susceptibility for some antibiotics in the ΔdnaKJ strain. PMID:27242140

  13. Chaperons expressions and search for new gravity-related genes in the embryos of crabs and amphibians

    NASA Astrophysics Data System (ADS)

    Gusev, O.; Kashiwagi, A.; Saigusa, M.

    Molecular mechanism of influence of gravity on living system is a subject of controversy for many years. Influence of gravity directly or indirectly affects to wide variety of biological processes, including biological clocks and general patterns in development of vertebrates and invertebrates. cDNA subtraction method was used for detection of the genes related to the hatching of the embryos semi-terrestrial crab Chiromantes haematocheir. Timing of the hatching of the embryos is highly synchronized with Moon phase and tides. While no new genes were found, we found that expression of chaperon hsp-90 increase in the embryos within two days before hatching, while expression of other stress proteins doesn't show any significant difference. Another model we used -- is a development of amphibian embryos. In order to clarify the effect of high gravity environment on development of Xenopus laevis, embryos on several developmental stages were subjected to the short-time high-gravity pulses (3G, 5G, and 9G). Analysis of stress-protein expression level and cDNA subtraction among high-gravity stressed embryos and control group revealed some changes in level of RNA expression of stress-proteins in experimental group. At the same time, we found two new genes expressed exclusively in the embryos under high gravity stress. The expression of the genes dramatically increased within several hours after the gravity stress, while the expression of the typical chaperons showed just slight difference. The genes expression pattern and its comparison with previously reported chaperons let us assume the presence physiological mechanism of specific gravity-stress response using previously unreported, special type of chaperons.

  14. Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

    PubMed Central

    Amm, Ingo; Norell, Derrick; Wolf, Dieter H.

    2015-01-01

    The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation. PMID:26466368

  15. Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

    PubMed Central

    Burmann, Björn M.; Holdbrook, Daniel A.; Callon, Morgane; Bond, Peter J.; Hiller, Sebastian

    2015-01-01

    The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function. PMID:25809264

  16. Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis.

    PubMed Central

    Kihlken, Margaret A; Leech, Andrew P; Le Brun, Nick E

    2002-01-01

    Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer. PMID:12238948

  17. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules.

    PubMed Central

    Vassilakos, A; Cohen-Doyle, M F; Peterson, P A; Jackson, M R; Williams, D B

    1996-01-01

    Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a molecular chaperone, based on indirect or correlative evidence. To examine calnexin's functions more directly, we reconstituted the assembly of class I histocompatibility molecules in the absence or presence of calnexin in Drosophila melanogaster cells. Calnexin enhanced the assembly of class I heavy chains with beta 2-microglobulin as much as 5-fold. The improved assembly appeared largely due to more efficient folding of heavy chains, as evidenced by increased reactivity with a conformation-sensitive monoclonal antibody and by a reduction in the level of aggregates. Similar findings were obtained in mouse or human cells when the interaction of calnexin with class I heavy chains was prevented by treatment with the oligosaccharide processing inhibitor castanospermine. The ability of calnexin to facilitate castanospermine. The ability of calnexin to facilitate heavy chain folding and to prevent the formation of aggregates provides compelling evidence that calnexin functions as a bona fide molecular chaperone. Images PMID:8612572

  18. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    NASA Astrophysics Data System (ADS)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  19. The co-chaperones Fkbp4/5 control Argonaute2 expression and facilitate RISC assembly

    PubMed Central

    Martinez, Natalia J.; Chang, Hao-Ming; Borrajo, Jacob de Riba; Gregory, Richard I.

    2013-01-01

    Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly. PMID:24049110

  20. Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

    PubMed Central

    Kaiser, Christian M.; Bujalowski, Paul J.; Ma, Liang; Anderson, John; Epstein, Henry F.; Oberhauser, Andres F.

    2012-01-01

    Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail. PMID:22824286

  1. The Hsp110 molecular chaperone stabilizes apolipoprotein B from endoplasmic reticulum-associated degradation (ERAD).

    PubMed

    Hrizo, Stacy L; Gusarova, Viktoria; Habiel, David M; Goeckeler, Jennifer L; Fisher, Edward A; Brodsky, Jeffrey L

    2007-11-01

    Apolipoprotein B (apoB) is the most abundant protein in low density lipoproteins and plays key roles in cholesterol homeostasis. The co-translational degradation of apoB is controlled by fatty acid levels in the endoplasmic reticulum (ER) and is mediated by the proteasome. To define the mechanism of apoB degradation, we employed a cell-free system in which proteasome-dependent degradation is recapitulated with yeast cytosol, and we developed an apoB yeast expression system. We discovered that a yeast Hsp110, Sse1p, associates with and stabilizes apoB, which contrasts with data indicating that select Hsp70s and Hsp90s facilitate apoB degradation. However, the Ssb Hsp70 chaperones have no effect on apoB turnover. To determine whether our results are relevant in mammalian cells, Hsp110 was overexpressed in hepatocytes, and enhanced apoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER-associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol. PMID:17823116

  2. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    PubMed Central

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  3. Molecular chaperones-related studies using latent stages of invertebrates exposed to space environment

    NASA Astrophysics Data System (ADS)

    Gusev, O. A.; Alexeev, V. R.; Sychev, V. N.; Okuda, T.; Saigusa, M.

    The latent stages of certain groups of invertebrates such as Artemia and Daphnia cyst Crustacea tuns of water bears Tardigrada are very perspective material for the investigation of the boundaries of the survival of the living organisms in the space environment While the number of authors showed that exposition the space flight causes the alteration in the survivability of the Artemia cysts there is no data about the changes in the stress response on the molecular level after short and long-termed space flight In this report we present preliminary results of the analysis of the expression of hsp90 chaperon in response to the heat shock in the larvae of the Artemia obtained from the cyst exposed to the real space flight onboard ISS for 1 and 6 month in the frame of the Aquarium program 2005-2006 and control ground group The perspectives of the usage of the molecular chaperons hsp in the studies for elucidation of the influence of the open space environment BIORISK and EXPOSE research programs on the immune response end general physiology of the invertebrates in their latent stages are discussed

  4. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

    PubMed

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M; Sim, Valerie L; Woodside, Michael T

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  5. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  6. Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

    PubMed

    Ghahghaei, Arezou; Valizadeh, Jafar; Nazari, Shahrzad; Ravandeh, Mehdi

    2014-06-01

    This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease. PMID:24599512

  7. Crystal Structures of Cisplatin Bound to a Human Copper Chaperone

    SciTech Connect

    Boal, Amie K.; Rosenzweig, Amy C.

    2010-08-16

    Copper trafficking proteins, including the chaperone Atox1 and the P{sub 1B}-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

  8. Generalized iterative annealing model for the action of RNA chaperones

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Thirumalai, D.

    2013-09-01

    As a consequence of the rugged landscape of RNA molecules their folding is described by the kinetic partitioning mechanism according to which only a small fraction (ϕF) reaches the folded state while the remaining fraction of molecules is kinetically trapped in misfolded intermediates. The transition from the misfolded states to the native state can far exceed biologically relevant time. Thus, RNA folding in vivo is often aided by protein cofactors, called RNA chaperones, that can rescue RNAs from a multitude of misfolded structures. We consider two models, based on chemical kinetics and chemical master equation, for describing assisted folding. In the passive model, applicable for class I substrates, transient interactions of misfolded structures with RNA chaperones alone are sufficient to destabilize the misfolded structures, thus entropically lowering the barrier to folding. For this mechanism to be efficient the intermediate ribonucleoprotein complex between collapsed RNA and protein cofactor should have optimal stability. We also introduce an active model (suitable for stringent substrates with small ϕF), which accounts for the recent experimental findings on the action of CYT-19 on the group I intron ribozyme, showing that RNA chaperones do not discriminate between the misfolded and the native states. In the active model, the RNA chaperone system utilizes chemical energy of adenosine triphosphate hydrolysis to repeatedly bind and release misfolded and folded RNAs, resulting in substantial increase of yield of the native state. The theory outlined here shows, in accord with experiments, that in the steady state the native state does not form with unit probability.

  9. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

    PubMed

    Costello, Shawn M; Plummer, Ashlee M; Fleming, Patrick J; Fleming, Karen G

    2016-08-16

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

  10. Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase.

    PubMed Central

    Kern, Renée; Malki, Abderrahim; Holmgren, Arne; Richarme, Gilbert

    2003-01-01

    Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function. PMID:12549977

  11. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  12. Heat shock proteins: molecular chaperones of protein biogenesis.

    PubMed Central

    Craig, E A; Gambill, B D; Nelson, R J

    1993-01-01

    Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins. PMID:8336673

  13. Mutational Analysis of Sse1 (Hsp110) Suggests an Integral Role for this Chaperone in Yeast Prion Propagation In Vivo

    PubMed Central

    Moran, Ciara; Kinsella, Gemma K.; Zhang, Zai-Rong; Perrett, Sarah; Jones, Gary W.

    2013-01-01

    The yeast Hsp110 chaperone Sse1 is a conserved protein that is a noncanonical member of the Hsp70 protein superfamily. Sse1 influences the cellular response to heat stress and has also been implicated in playing a role in the propagation of prions in yeast. Sse1 can seemingly exert its effects in vivo through direct or indirect actions by influencing the nucleotide exchange activity of canonical cytosolic Hsp70s. Using a genetic screen based on the inability to propagate the yeast [PSI+] prion, we have identified 13 new Sse1 mutants that are predicted to alter chaperone function through a variety of different mechanisms. Not only are these new Sse1 mutants altered in the ability to propagate and cure yeast prions but also to varying degrees in the ability to grow at elevated temperatures. The expression levels of chaperone proteins known to influence yeast prion propagation are unaltered in the Sse1 mutants, suggesting that the observed phenotypic effects are caused by direct functional alterations in these mutants. Mapping the location of the mutants onto the Sse1 crystal structure suggests that more than one functional alteration in Sse1 may result in changes in prion propagation and ability to function at elevated temperatures. All Sse1 mutants isolated provide essential functions in the cell under normal growth conditions, further demonstrating that essential chaperone functions in vivo can to some degree at least be detached from those related to propagation of prions. Our results suggest that Sse1 can influence prion propagation through a variety of different mechanisms. PMID:23797105

  14. Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

    PubMed Central

    Thomson, Nicholas M.; Saika, Azusa; Ushimaru, Kazunori; Sangiambut, Smith; Tsuge, Takeharu; Summers, David K.

    2013-01-01

    The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. PMID:23335776

  15. Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

    PubMed

    Haga, Asami; Okamoto, Tomoya; Yamada, Shintaroh; Kubota, Toshihiko; Sanpei, Ann; Takahashi, Shota; Nakayama, Masahiro; Nagai, Miki; Otaka, Michiro; Miyazaki, Toshio; Nunomura, Wataru; Grave, Ewa; Itoh, Hideaki

    2013-09-01

    In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70. PMID:23687308

  16. Chaperone-assisted excisive recombination, a solitary role for DnaJ (Hsp40) chaperone in lysogeny escape.

    PubMed

    Champ, Stéphanie; Puvirajesinghe, Tania M; Perrody, Elsa; Menouni, Rachid; Genevaux, Pierre; Ansaldi, Mireille

    2011-11-11

    Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development. PMID:21908845

  17. The co-chaperone CHIP is induced in various stresses and confers protection to cells.

    PubMed

    Dikshit, Priyanka; Jana, Nihar Ranjan

    2007-06-01

    The C-terminus of Hsp70 interacting protein (CHIP) is being considered to be a cellular quality control E3 ubiquitin ligase because of its ability to degrade misfolded proteins in association with heat shock chaperones. The neuroprotective role of CHIP also has been implicated in several familial neurodegenerative diseases including polyglutamine diseases. However, the regulation of the expression of CHIP under different stress conditions and its protective role thereon is unknown. Here we have shown that the mRNA level of CHIP is significantly increased in the cells exposed to oxidative, endoplasmic reticulum and proteasomal stress. CHIP also protected from various stress-induced cell death. Finally, we have demonstrated upregulation of CHIP mRNA levels in the expanded polyglutamine protein expressing cells. Our result suggests that the upregulation of CHIP under various stress environments is an adaptive response of the cells to deal with the excess burden of misfolded protein. PMID:17442270

  18. The role of HSP70 and its co-chaperones in protein misfolding, aggregation and disease.

    PubMed

    Duncan, Emma J; Cheetham, Michael E; Chapple, J Paul; van der Spuy, Jacqueline

    2015-01-01

    Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein folding, quality control and function. In particular, the HSP70 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and its co-chaperones have been recognised as potent modulators of inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. In has become evident that the HSP70 chaperone machine functions not only in folding, but also in proteasome mediated degradation of neurodegenerative disease proteins. Thus, there has been a great deal of interest in the potential manipulation of molecular chaperones as a therapeutic approach for many neurodegenerations. Furthermore, mutations in several HSP70 co-chaperones and putative co-chaperones have been identified as causing inherited neurodegenerative and cardiac disorders, directly linking the HSP70 chaperone system to human disease. PMID:25487025

  19. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    PubMed Central

    Lawless, Nathan; Blacklock, Kristin; Berrigan, Elizabeth; Verkhivker, Gennady

    2013-01-01

    A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4) kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock) kinase from the system during client loading (release) stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery. PMID:24287464

  20. Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli.

    PubMed

    Peng, Shuaiying; Chu, Zhongmei; Lu, Jianfeng; Li, Dongxiao; Wang, Yonghong; Yang, Shengli; Zhang, Yi

    2016-05-01

    The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that co-expression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA. PMID:26862080

  1. BtcA, A Class IA Type III Chaperone, Interacts with the BteA N-Terminal Domain through a Globular/Non-Globular Mechanism

    PubMed Central

    Guttman, Chen; Davidov, Geula; Yahalom, Adi; Shaked, Hadassa; Kolusheva, Sofiya; Bitton, Ronit; Barber-Zucker, Shiran; Chill, Jordan H.; Zarivach, Raz

    2013-01-01

    Bordetella pertussis, the etiological agent of “whooping cough” disease, utilizes the type III secretion system (T3SS) to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry. PMID:24312558

  2. Molecular characterization of two novel molecular chaperones in bacterial-challenged Apostichopus japonicus.

    PubMed

    Wang, Haihong; Shao, Yina; Zhang, Weiwei; Li, Chenghua; Lv, Zhimeng; Jin, Chunhua

    2015-10-01

    Molecular chaperones of 78 kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) are involved in protein folding and assembly in the endoplasmic reticulum (ER). Increasing evidences also suggest that these two molecules play an important role in immune response. In the present study, we cloned and characterized GRP78 and PDI genes from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjGRP78 and AjPDI, respectively). The AjGRP78 cDNA was of 2355bp including an open reading frame (ORF) of 2013 bp encoding a protein of 670 amino acids with three heat shock protein 70 (HSP70) family signatures. AjGRP78 contained a 23-amino acid signal peptide at the N-terminus and a HDEL motif at the C-terminus, which supported the location of the protein in the ER. The full length cDNA of AjPDI was of 1893 bp with a 5' untranslated region (UTR) of 153 bp, a 3' UTR of 228 bp and an ORF of 1512 bp encoding a protein of 503 amino acids. A 17-amino acid signal peptide, two thioredoxin domains with two active sites of CGHC, and KDEL retention signal were totally conserved in the deduced amino acid of AjPDI. Phylogenic analysis and multiple alignments have shown that both genes shared remarkably higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates, further supporting that the two proteins were novel members of molecular chaperone family. Spatial expression analysis revealed that AjGRP78 mRNA transcripts were dominantly expressed in the tentacle, while AjPDI mRNA levels were abundant in the muscle, intestine and respiratory trees. For Vibrio splendidus challenged sea cucumber, the peak expression of AjGRP78 and AjPDI mRNAs in coelomocytes were detected at 24h with 1.73-fold increase and at 6h with 1.83-fold increase compared with the control group, respectively. Similarly, a significant increase in the relative mRNA levels of AjGRP78 and AjPDI was also identified in 1 μg mL(-1

  3. Chaperone proteins and brain tumors: Potential targets and possible therapeutics1

    PubMed Central

    Graner, Michael W.; Bigner, Darell D.

    2005-01-01

    Chaperone proteins are most notable for the proteo- and cyotoprotective capacities they afford during cellular stress. Under conditions of cellular normalcy, chaperones still play integral roles in the folding of nascent polypeptides into functional entities, in assisting in intracellular/intraorganellar transport, in assembly and maintenance of multi-subunit protein complexes, and in aiding and abetting the degradation of senescent proteins. Tumors frequently have relatively enhanced needs for chaperone number and activity because of the stresses of rapid proliferation, increased metabolism, and overall genetic instability. Thus, it may be possible to take advantage of this reliance that tumor cells have on chaperones by pharmacologic and biologic means. Certain chaperones are abundant in the brain, which implies important roles for them. While it is presumed that the requirements of brain tumors for chaperone proteins are similar to those of any other cell type, tumor or otherwise, very little inquiry has been directed at the possibility of using chaperone proteins as therapeutic targets or even as therapeutic agents against central nervous system malignancies. This review highlights some of the research on the functions of chaperone proteins, on what can be done to modify those functions, and on the physiological responses that tumors and organisms can have to chaperone-targeted or chaperone-based therapies. In particular, this review will also underscore areas of research where brain tumors have been part of the field, although in general those instances are few and far between. This relative dearth of research devoted to chaperone protein targets and therapeutics in brain tumors reveals much untrodden turf to explore for potential treatments of these dreadfully refractive diseases. PMID:16053701

  4. Genomic organization of ATOX1, a human copper chaperone

    PubMed Central

    Liu, Po-Ching; Koeller, David M; Kaler, Stephen G

    2003-01-01

    Background Copper is an essential trace element that plays a critical role in the survival of all living organisms. Menkes disease and occipital horn syndrome (OHS) are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A) that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network. Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways. Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein. Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mobr), a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported. Results We determined the genomic structure of ATOX1. The gene contains 4 exons spanning a genomic distance of approximately 16 kb. The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3. We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA. We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found. Conclusions In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport. PMID:12594858

  5. A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

    PubMed

    Hamdane, Djemel; Velours, Christophe; Cornu, David; Nicaise, Magali; Lombard, Murielle; Fontecave, Marc

    2016-07-27

    Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the

  6. The small heat shock proteins family: the long forgotten chaperones.

    PubMed

    Garrido, C; Paul, C; Seigneuric, R; Kampinga, H H

    2012-10-01

    Small heat shock proteins are a rather heterogeneous family of ATP-independent chaperones, some of which have been proven to block protein aggregation and help the cells to survive stressful conditions. Although much less studied than high molecular weight HSPs like HSP70/HSPA or HSP90/HSPC, their implication in physio-pathological processes and human diseases is now well evidenced, as it will be discussed in the different reviews of this special issue. In this mini-review we will just present a general introduction about the small heat shock proteins family. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. PMID:22449631

  7. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    PubMed

    Lavado-Roldán, Angela; Fernández-Chacón, Rafael

    2016-07-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  8. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2

    PubMed Central

    2016-01-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  9. The Role of Bacterial Chaperones in the Circulative Transmission of Plant Viruses by Insect Vectors

    PubMed Central

    Kliot, Adi; Ghanim, Murad

    2013-01-01

    Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed. PMID:23783810

  10. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    PubMed Central

    Kaushik, Susmita; Cuervo, Ana Maria

    2015-01-01

    Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LD) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation via CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LD. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs lead to reduced association of ATGL and macrolipophagy-related proteins with LD and the subsequent decrease in lipid oxidation and accumulation of LD. We propose a role of CMA in LD biology and in the maintenance of lipid homeostasis. PMID:25961502

  11. The Role of Copper Chaperone Atox1 in Coupling Redox Homeostasis to Intracellular Copper Distribution.

    PubMed

    Hatori, Yuta; Lutsenko, Svetlana

    2016-01-01

    Human antioxidant protein 1 (Atox1) is a small cytosolic protein with an essential role in copper homeostasis. Atox1 functions as a copper carrier facilitating copper transfer to the secretory pathway. This process is required for activation of copper dependent enzymes involved in neurotransmitter biosynthesis, iron efflux, neovascularization, wound healing, and regulation of blood pressure. Recently, new cellular roles for Atox1 have emerged. Changing levels of Atox1 were shown to modulate response to cancer therapies, contribute to inflammatory response, and protect cells against various oxidative stresses. It has also become apparent that the activity of Atox1 is tightly linked to the cellular redox status. In this review, we summarize biochemical information related to a dual role of Atox1 as a copper chaperone and an antioxidant. We discuss how these two activities could be linked and contribute to establishing the intracellular copper balance and functional identity of cells during differentiation. PMID:27472369

  12. Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

    NASA Technical Reports Server (NTRS)

    Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

    2003-01-01

    Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

  13. [CHAPERONES FUNCTION HSP60 AND HSP90 AND THEIR ROLE IN CARDIAC PATHOLOGY].

    PubMed

    Kazimirko, V K; Kutovoy, V V; Bobyk, V I; Kozak, I O; Ivanitskaya, L M; Dubkova, A G; Silanteva, T S

    2014-01-01

    In review provides information about the function oft the body of chaperones and their role in the development of pathological processes, including--atherosclerosis and coronary heart disease. Marked comminications systems chaperones to the immune and endocrine systems, and inflammation. PMID:26492771

  14. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  15. Targeting Molecular Chaperones for the Treatment of Cystic Fibrosis: Is It a Viable Approach?

    PubMed

    Heard, Ashley; Thompson, Jake; Carver, Jessica; Bakey, Michelle; Wang, X Robert

    2015-01-01

    Cystic Fibrosis (CF) is largely caused by protein misfolding and the loss of function of a plasma membrane anion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common CF-causing mutation, F508del, leads to severe conformational defect in CFTR. The cellular chaperone machinery plays an important role in CFTR biogenesis and quality control. Multiple attempts have been made to improve the cell surface functional expression of the mutant CFTR by modulating the expression of components of the cellular chaperone machinery. The efficacy of such an approach has been low largely due to the severe intrinsic folding defects of the F508del CFTR. Moreover, the impact of chaperone perturbation on the chaperone machinery itself and on other physiologically important proteins might lead to potentially severe side effects. Approaches aimed at disrupting chaperone-CFTR interactions show greater efficacy, and are compatible with small-molecule drug discovery and gene therapy. Combination between chaperone modulators and F508del correctors might further enhance potency and specificity. As molecular chaperones play important roles in regulating inflammation and immunity, they can be potential targets for controlling airway infection and inflammation in patients. If such effects can be synergized with chaperone-mediated regulation of CFTR biogenesis and quality control, more efficacious therapeutics will be developed to combat CF lung disease. PMID:25981601

  16. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation

    NASA Astrophysics Data System (ADS)

    Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Månsson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2016-03-01

    It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.

  17. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation

    PubMed Central

    Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Månsson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2016-01-01

    It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation. PMID:27009901

  18. Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.

    PubMed

    Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P; Abell, Benjamin M; Nabok, Alexei

    2011-07-20

    This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. PMID:21767504

  19. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    DOE PAGESBeta

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; Salamone, Stéphane; Christensen, Stig H.; Read, Randy J.; Moriarty, Nigel W.; Jensen, Henrik H.; Deane, Janet E.

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe amore » new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.« less

  20. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    SciTech Connect

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; Salamone, Stéphane; Christensen, Stig H.; Read, Randy J.; Moriarty, Nigel W.; Jensen, Henrik H.; Deane, Janet E.

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.

  1. Structural basis underlying viral hijacking of a histone chaperone complex.

    PubMed

    Huang, Hongda; Deng, Zhong; Vladimirova, Olga; Wiedmer, Andreas; Lu, Fang; Lieberman, Paul M; Patel, Dinshaw J

    2016-01-01

    The histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. Here we report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through non-conserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host-antiviral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an antiviral histone chaperone to promote viral latency and cellular immortalization. PMID:27581705

  2. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase

    PubMed Central

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-01-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. PMID:23158495

  3. [Unfolding chaperone as a prion protein relating molecule].

    PubMed

    Hachiya, Naomi S; Sakasegawa, Yuji; Kaneko, Kiyotoshi

    2003-11-01

    Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent. PMID:15152473

  4. Anticancer Gold(III) Porphyrins Target Mitochondrial Chaperone Hsp60.

    PubMed

    Hu, Di; Liu, Yungen; Lai, Yau-Tsz; Tong, Ka-Chung; Fung, Yi-Man; Lok, Chun-Nam; Che, Chi-Ming

    2016-01-22

    Identification of the molecular target(s) of anticancer metal complexes is a formidable challenge since most of them are unstable toward ligand exchange reaction(s) or biological reduction under physiological conditions. Gold(III) meso-tetraphenylporphyrin (gold-1 a) is notable for its high stability in biological milieux and potent in vitro and in vivo anticancer activities. Herein, extensive chemical biology approaches employing photo-affinity labeling, click chemistry, chemical proteomics, cellular thermal shift, saturation-transfer difference NMR, protein fluorescence quenching, and protein chaperone assays were used to provide compelling evidence that heat-shock protein 60 (Hsp60), a mitochondrial chaperone and potential anticancer target, is a direct target of gold-1 a in vitro and in cells. Structure-activity studies with a panel of non-porphyrin gold(III) complexes and other metalloporphyrins revealed that Hsp60 inhibition is specifically dependent on both the gold(III) ion and the porphyrin ligand. PMID:26663758

  5. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    PubMed

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. PMID:23158495

  6. Co-chaperone CHIP promotes aggregation of ataxin-1.

    PubMed

    Choi, Jung Young; Ryu, Jeong Hee; Kim, Hyo-Sun; Park, Sung Goo; Bae, Kwang-Hee; Kang, Sunghyun; Myung, Pyung Keun; Cho, Sayeon; Park, Byoung Chul; Lee, Do Hee

    2007-01-01

    Recent studies demonstrated that co-chaperone/E3 ligase CHIP (C-terminus of hsp70-interacting protein) mediates the ubiquitylation and suppresses the aggregation of polyglutamine (polyQ) proteins, such as huntingtin or ataxin-3. In this study, we investigated the effects of CHIP on the degradation of another polyQ protein ataxin-1. Interestingly CHIP associates not only with the polyQ-expanded ataxin-1 but also with the normal ataxin-1. Moreover, by enhancing ataxin-1 ubiquitylation, CHIP over-expression leads to a reduction in the solubility of ataxin-1 and thus increases the aggregate formation, especially that of polyQ-expanded ataxin-1. Domain analysis revealed that the TPR domain is required for the promotion of aggregation. By contrast, other co-chaperones or E3 ligases, such as BAG-1 or parkin, did not show similar effects on the aggregation of ataxin-1. Importantly, the effect of CHIP is impaired by the mutation of Ser776 of ataxin-1 whose phosphorylation is crucial for ataxin-1 aggregation. Our findings suggest that the role of CHIP in aggregation of polyQ proteins greatly varies depending on the context of full-length polyQ proteins. PMID:17127076

  7. Classification of chemical chaperones based on their effect on protein folding landscapes.

    PubMed

    Dandage, Rohan; Bandyopadhyay, Anannya; Jayaraj, Gopal Gunanathan; Saxena, Kanika; Dalal, Vijit; Das, Aritri; Chakraborty, Kausik

    2015-03-20

    Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems. PMID:25493352

  8. The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation.

    PubMed

    Entzminger, Kevin C; Chang, Christine; Myhre, Ryan O; McCallum, Katie C; Maynard, Jennifer A

    2012-06-19

    The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence. PMID:22650963

  9. Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation

    PubMed Central

    Kłosowska, Agnieszka; Chamera, Tomasz; Liberek, Krzysztof

    2016-01-01

    Hsp104 disaggregase provides thermotolerance in yeast by recovering proteins from aggregates in cooperation with the Hsp70 chaperone. Protein disaggregation involves polypeptide extraction from aggregates and its translocation through the central channel of the Hsp104 hexamer. This process relies on adenosine triphosphate (ATP) hydrolysis. Considering that Hsp104 is characterized by low affinity towards ATP and is strongly inhibited by adenosine diphosphate (ADP), we asked how Hsp104 functions at the physiological levels of adenine nucleotides. We demonstrate that physiological levels of ADP highly limit Hsp104 activity. This inhibition, however, is moderated by the Hsp70 chaperone, which allows efficient disaggregation by supporting Hsp104 binding to aggregates but not to non-aggregated, disordered protein substrates. Our results point to an additional level of Hsp104 regulation by Hsp70, which restricts the potentially toxic protein unfolding activity of Hsp104 to the disaggregation process, providing the yeast protein-recovery system with substrate specificity and efficiency in ATP consumption. DOI: http://dx.doi.org/10.7554/eLife.15159.001 PMID:27223323

  10. Modeling and analysis of prion dynamics in the presence of a chaperone.

    PubMed

    Kumar, Rajiv; Murali, Padma

    2008-05-01

    Prions are infectious agents and are polymers called PrP(Sc)-Prion protein scrapies, of a normal protein, a monomer called PrP(c)-Prion protein cellular. These PrP(Sc)s cause TSEs-transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE) in cattle, scrapies in sheep, Kuru and Creutzfeld-Jacob diseases in humans. Cellular molecular chaperones, which are ubiquitous, stress-induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein-misfolding diseases. In this work, we propose a model for the replication of prions by nucleated polymerization in the presence of a chaperone. According to this model, the biological processes of coagulation, splitting and the inhibitory effects of the chaperone can be described by a coupled system consisting of ordinary differential equations and a partial differential equation. The model is converted into a system of ordinary differential equations and the equilibrium points are computed and their stability is studied. We give a numerical simulation of the model and we find that a disease free state can be achieved in the presence of a chaperone. The duration of the disease free state is found to increase with the amount of chaperone and this amount of chaperone can be computed from the model. PMID:18362035

  11. Molecular chaperones: guardians of the proteome in normal and disease states

    PubMed Central

    Jeng, Wilson; Lee, Sukyeong; Sung, Nuri; Lee, Jungsoon; Tsai, Francis T.F.

    2015-01-01

    Proteins must adopt a defined three-dimensional structure in order to gain functional activity, or must they? An ever-increasing number of intrinsically disordered proteins and amyloid-forming polypeptides challenge this dogma. While molecular chaperones and proteases are traditionally associated with protein quality control inside the cell, it is now apparent that molecular chaperones not only promote protein folding in the “forward” direction by facilitating folding and preventing misfolding and aggregation, but also facilitate protein unfolding and even disaggregation resulting in the recovery of functional protein from aggregates. Here, we review our current understanding of ATP-dependent molecular chaperones that harness the energy of ATP binding and hydrolysis to fuel their chaperone functions. An emerging theme is that most of these chaperones do not work alone, but instead function together with other chaperone systems to maintain the proteome. Hence, molecular chaperones are the major component of the proteostasis network that guards and protects the proteome from damage. Furthermore, while a decline of this network is detrimental to cell and organismal health, a controlled perturbation of the proteostasis network may offer new therapeutic avenues against human diseases. PMID:26918154

  12. M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production

    PubMed Central

    Feng, Jiaying; Gong, Danyang; Fu, Xudong; Wu, Ting-ting; Wang, Jane; Chang, Jennifer; Zhou, Jingting; Lu, Gang; Wang, Yibin; Sun, Ren

    2015-01-01

    Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways. PMID:26615759

  13. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    PubMed

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI) network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics. PMID:24244114

  14. Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18

    PubMed Central

    Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis

    2015-01-01

    Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662

  15. Molecular Chaperones as Rational Drug Targets for Parkinson’s Disease Therapeutics

    PubMed Central

    Kalia, S.K.; Kalia, L.V.; McLean, P.J.

    2012-01-01

    Parkinson’s disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson’s disease and of genetic susceptibility variants for idiopathic Parkinson’s disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the co-chaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson’s disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson’s disease. PMID:20942788

  16. In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

    PubMed Central

    Andley, Usha P.; Malone, James P.; Townsend, R. Reid

    2014-01-01

    αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin. PMID:24760011

  17. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  18. The Spliceosome: The Ultimate RNA Chaperone and Sculptor.

    PubMed

    Papasaikas, Panagiotis; Valcárcel, Juan

    2016-01-01

    The spliceosome, one of the most complex machineries of eukaryotic cells, removes intronic sequences from primary transcripts to generate functional messenger and long noncoding RNAs (lncRNA). Genetic, biochemical, and structural data reveal that the spliceosome is an RNA-based enzyme. Striking mechanistic and structural similarities strongly argue that pre-mRNA introns originated from self-catalytic group II ribozymes. However, in the spliceosome, protein components organize and activate the catalytic-site RNAs, and recognize and pair together splice sites at intron boundaries. The spliceosome is a dynamic, reversible, and flexible machine that chaperones small nuclear (sn) RNAs and a variety of pre-mRNA sequences into conformations that enable intron removal. This malleability likely contributes to the regulation of alternative splicing, a prevalent process contributing to cell differentiation, homeostasis, and disease. PMID:26682498

  19. pH-Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases.

    PubMed

    Mena-Barragán, Teresa; Narita, Aya; Matias, Dino; Tiscornia, Gustavo; Nanba, Eiji; Ohno, Kousaku; Suzuki, Yoshiyuki; Higaki, Katsumi; Garcia Fernández, José Manuel; Ortiz Mellet, Carmen

    2015-09-28

    A general approach is reported for the design of small-molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self-inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH-responsive glycomimetics targeting human glucocerebrosidase or α-galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed. PMID:26386364

  20. Chemical chaperones assist intracellular folding to buffer mutational variations

    PubMed Central

    Bandyopadhyay, Anannya; Saxena, Kanika; Kasturia, Neha; Dalal, Vijit; Bhatt, Niraj; Rajkumar, Asher; Maity, Shuvadeep; Sengupta, Shantanu; Chakraborty, Kausik

    2012-01-01

    Hidden genetic variations harbor potential for the evolution of new traits. Molecular chaperones, that assist protein folding, may conceal genetic variations in protein coding regions. Here, we investigate if the chemical milieu of cells has the potential to alleviate intracellular protein folding; potentially implicating a role of osmolytes in concealing genetic variations. Using the model osmolyte TMAO, we uncover that it can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. Strikingly, we delineate different osmolytes to have a unique spectrum of buffered-mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering. PMID:22246401

  1. The nucleotide exchange factors of Hsp70 molecular chaperones

    PubMed Central

    Bracher, Andreas; Verghese, Jacob

    2015-01-01

    Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell. PMID:26913285

  2. Multiple functions of the histone chaperone Jun dimerization protein 2.

    PubMed

    Tsai, Ming-Ho; Wuputra, Kenly; Lin, Yin-Chu; Lin, Chang-Shen; Yokoyama, Kazunari K

    2016-09-30

    The Jun dimerization protein 2 (JDP2) is part of the family of stress-responsible transcription factors such as the activation protein-1, and binds the 12-O-tetradecanoylphorbol-13-acetateresponse element and the cAMP response element. It also plays a role as a histone chaperone and participates in diverse processes, such as cell-cycle arrest, cell differentiation, apoptosis, senescence, and metastatic spread, and functions as an oncogene and anti-oncogene, and as a cellular reprogramming factor. However, the molecular mechanisms underlying these multiple functions of JDP2 have not been clarified. This review summarizes the structure and function of JDP2, highlighting the specific role of JDP2 in cellular-stress regulation and prevention. PMID:27041241

  3. Antarctic Krill 454 Pyrosequencing Reveals Chaperone and Stress Transcriptome

    PubMed Central

    Clark, Melody S.; Thorne, Michael A. S.; Toullec, Jean-Yves; Meng, Yan; Guan, Le Luo; Peck, Lloyd S.; Moore, Stephen

    2011-01-01

    Background The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. Methodology/Principal Findings The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies. Conclusions This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical “stress proteins”, such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of “stress” genes for studies understanding marine

  4. Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide

    PubMed Central

    Macri, Christophe; Wang, Fengjuan; Tasset, Inmaculada; Schall, Nicolas; Page, Nicolas; Briand, Jean-Paul; Cuervo, Ana Maria; Muller, Sylviane

    2015-01-01

    The P140 peptide, a 21-mer linear peptide (sequence 131–151) generated from the spliceosomal SNRNP70/U1–70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired. PMID:25719862

  5. Oxidative switches in functioning of mammalian copper chaperone Cox17

    PubMed Central

    Voronova, Anastassia; Meyer-Klaucke, Wolfram; Meyer, Thomas; Rompel, Annette; Krebs, Bernt; Kazantseva, Jekaterina; Sillard, Rannar; Palumaa, Peep

    2007-01-01

    Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox170S–S binds co-operatively to four Cu+; Cox172S–S, with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox173S–S, with three disulfide bridges, does not bind to any metal ions. The Em (midpoint redox potential) values for two redox couples of Cox17, Cox173S–S↔Cox172S–S (Em1) and Cox172S–S↔Cox170S–S (Em2), were determined to be −197 mV and −340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox170S-S and Cox172S–S, which is slightly shifted towards Cox170S-S. In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox172S–S, enabling retention of Cox172S–S in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox172S–S, capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper–thiolate cluster, which may provide safe storage of an excess of copper ions. PMID:17672825

  6. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  7. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    PubMed

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool. PMID:27430516

  8. Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival‡

    PubMed Central

    Shi, Lei; Ravikumar, Vaishnavi; Derouiche, Abderahmane; Macek, Boris; Mijakovic, Ivan

    2016-01-01

    In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis. PMID:27148221

  9. [Mutation clpA::kan in gene encoding the chaperone of Hsp100-family decreases DnaK-dependent refolding efficiency of proteins in Escherichia coli cells].

    PubMed

    Kotova, V Iu; Manukhov, i V; Mel'kina, O E; Zavil'gel'skiĭ, G B

    2008-01-01

    The rate and level of DnaK-dependent refolding of the thermoinactivated Vibrio fischeri luciferase were considerably lower in clpA mutant (clpA::kan) then in wild type cells. The decline of level of refolding makes progress with the increase of thermoinactivation time of enzyme. The mutation in clpP gene has no influence on kinetic and level of luciferase refolding. It was shown that the approximately equal amounts of the DnaKJE chaperone are synthesized under "heat shock" induction in E. coli clpA+ and E. coli clpA::kan cells. We suppose that the chaperone ClpA (like homological chaperone ClpB) is involved in the disaggregation process of denaturized proteins and that results to the increase of refolding efficacy. This in vivo phenomenon occurs only under long time incubation of cells at a high temperature, i.e. when protein aggregates of large size poorly refoldable by the DnaKJE system are formed. PMID:19140322

  10. Single Amino Acid Deletion in Kindlin-1 Results in Partial Protein Degradation Which Can Be Rescued by Chaperone Treatment.

    PubMed

    Maier, Kristin; He, Yinghong; Esser, Philipp R; Thriene, Kerstin; Sarca, Daniela; Kohlhase, Jürgen; Dengjel, Jörn; Martin, Ludovic; Has, Cristina

    2016-05-01

    Kindler syndrome, a distinct type of epidermolysis bullosa, is a rare disorder caused by mutations in FERMT1, encoding kindlin-1. Most FERMT1 mutations lead to premature termination codons and absence of kindlin-1. Here we investigated the molecular and cellular consequences of a naturally occurring FERMT1 mutation, c.299_301del resulting in a single amino acid deletion, p.R100del. The mutation led to a 50% reduction of FERMT1 mRNA and 90% reduction of kindlin-1 protein in keratinocytes derived from the patient, as compared with control cells. The misfolded p.R100del kindlin-1 mutant was lysosomally degraded and launched a homeostatic unfolded protein response. Sodium-phenylbutyrate significantly increased kindlin-1 mRNA and protein levels and the area of mutant cells, acting as a chemical chaperone and probably also as a histone deacetylase inhibitor. In a recombinant system, low levels of wild-type or p.R100del mutant kindlin-1 were sufficient to improve the cellular phenotype in respect of spreading and proliferation as compared with kindlin-1 negative keratinocytes. The study of this hypomorphic mutation provides evidence that low amounts of kindlin-1 are sufficient to improve the epidermal architecture and Kindler syndrome cellular phenotype and proposes a personalized chaperone therapy for the patient. PMID:26827766

  11. Macrophage Migration Inhibitory Factor (MIF) as a Chaperone Inhibiting Accumulation of Misfolded SOD1

    PubMed Central

    Israelson, Adrian; Ditsworth, Dara; Sun, Shuying; Song, SungWon; Liang, Jason; Hruska-Plochan, Marian; McAlonis-Downes, Melissa; Abu-Hamad, Salah; Zoltsman, Guy; Shani, Tom; Maldonado, Marcus; Bui, Anh; Navarro, Michael; Zhou, Huilin; Marsala, Martin; Kaspar, Brian K.; Da Cruz, Sandrine; Cleveland, Don W.

    2015-01-01

    Summary Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in non-neuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity. PMID:25801706

  12. Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek†

    PubMed Central

    Nuccio, Sean-Paul; Bäumler, Andreas J.

    2007-01-01

    Summary: Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed α-, β-, γ-, κ-, π-, and σ-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups. PMID:18063717

  13. The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

    PubMed Central

    Bowman, Andrew; Hammond, Colin M.; Stirling, Andrew; Ward, Richard; Shang, Weifeng; El-Mkami, Hassane; Robinson, David A.; Svergun, Dmitri I.; Norman, David G.; Owen-Hughes, Tom

    2014-01-01

    NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism. PMID:24688059

  14. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    SciTech Connect

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  15. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    DOE PAGESBeta

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  16. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    SciTech Connect

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  17. The Hsp90 molecular chaperone complex regulates maltose induction and stability of the Saccharomyces MAL gene transcription activator Mal63p.

    PubMed

    Bali, Mehtap; Zhang, Bin; Morano, Kevin A; Michels, Corinne A

    2003-11-28

    Induction of the Saccharomyces MAL structural genes encoding maltose permease and maltase requires the MAL activator, a DNA-binding transcription activator. Genetic analysis of MAL activator mutations suggested that protein folding and stability play an important role in MAL activator regulation and led us to explore the role of the Hsp90 molecular chaperone complex in the regulation of the MAL activator. Strains carrying mutations in genes encoding components of the Hsp90 chaperone complex, hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta, are defective for maltase induction and exhibit significantly reduced growth rates on media containing a limiting concentration of maltose (0.05%). This growth defect is suppressed by providing maltose in excess. Using epitope-tagged alleles of the MAL63 MAL activator, we showed that Mal63p levels are drastically reduced following depletion of cellular Hsp90. Overexpression ( approximately 3-fold) of Mal63p in the hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta strains suppresses their Mal- growth phenotype, suggesting that Mal63p levels are limiting for maltose utilization in strains with abrogated Hsp90 activity. Consistent with this, the half-life of Mal63p is significantly shorter in the hsc82 Delta cpr7 Delta strain (reduced about 6-fold) and modestly affected in the Hsp90-ts strain (reduced about 2-fold). Most importantly, triple hemagglutinin-tagged Mal63p protein is found in association with Hsp90 as demonstrated by co-immunoprecipitation. Taken together, these results identify the inducible MAL activator as a client protein of the Hsp90 molecular chaperone complex and point to a critical role for chaperone function in alternate carbon source utilization in Saccharomyces cerevisiae. PMID:14500708

  18. Molecular Chaperones in Parkinson’s Disease – Present and Future

    PubMed Central

    Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J.

    2011-01-01

    Parkinson’s disease, like many other neurodegenerative disorders, is characterized by the progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. The cascade by which the small synaptic protein α-synuclein misfolds to form distinctive protein aggregates, termed Lewy bodies and Lewy neurites, has been the subject of intensive research for more than a decade. Genetic and pathological studies in Parkinson’s disease patients as well as experimental studies in disease models have clearly established altered protein metabolism as a key element in the pathogenesis of Parkinson’s disease. Alterations in protein metabolism include misfolding and aggregation, post-translational modification and dysfunctional degradation of cytotoxic protein species. Protein folding and re-folding are both mediated by a highly conserved network of molecules, called molecular chaperones and co-chaperones. In addition to the regulatory role in protein folding, molecular chaperone function is intimately associated with pathways of protein degradation, such as the ubiquitin-proteasome system and the autophagy-lysosomal pathway, to effectively remove irreversibly misfolded proteins. Because of the central role of molecular chaperones in maintaining protein homeostasis, we herein review our current knowledge on the involvement of molecular chaperones and co-chaperones in Parkinson’s disease. We further discuss the capacity of molecular chaperones to prevent or modulate neurodegeneration, an important concept for future neuroprotective strategies and summarize the current progress in preclinical studies in models of Parkinson’s disease and other neurodegenerative disorders. Finally we include a discussion on the future potential of using molecular chaperones as a disease modifying therapy. PMID:22279517

  19. Investigation of original multivalent iminosugars as pharmacological chaperones for the treatment of Gaucher disease.

    PubMed

    Laigre, Eugénie; Hazelard, Damien; Casas, Josefina; Serra-Vinardell, Jenny; Michelakakis, Helen; Mavridou, Irene; Aerts, Johannes M F G; Delgado, Antonio; Compain, Philippe

    2016-06-24

    Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a lysosome-targeting morpholine unit into iminosugar cluster structures has no significant impact on the chaperone activity on Gaucher cells. PMID:27063390

  20. Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones

    PubMed Central

    Narayan, Priyanka; Meehan, Sarah; Carver, John A.; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer’s disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment. PMID:23106396

  1. Organismal proteostasis: role of cell-nonautonomous regulation and transcellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Morimoto, Richard I.

    2014-01-01

    Protein quality control is essential in all organisms and regulated by the proteostasis network (PN) and cell stress response pathways that maintain a functional proteome to promote cellular health. In this review, we describe how metazoans employ multiple modes of cell-nonautonomous signaling across tissues to integrate and transmit the heat-shock response (HSR) for balanced expression of molecular chaperones. The HSR and other cell stress responses such as the unfolded protein response (UPR) can function autonomously in single-cell eukaryotes and tissue culture cells; however, within the context of a multicellular animal, the PN is regulated by cell-nonautonomous signaling through specific sensory neurons and by the process of transcellular chaperone signaling. These newly identified forms of stress signaling control the PN between neurons and nonneuronal somatic tissues to achieve balanced tissue expression of chaperones in response to environmental stress and to ensure that metastable aggregation-prone proteins expressed within any single tissue do not generate local proteotoxic risk. Transcellular chaperone signaling leads to the compensatory expression of chaperones in other somatic tissues of the animal, perhaps preventing the spread of proteotoxic damage. Thus, communication between subcellular compartments and across different cells and tissues maintains proteostasis when challenged by acute stress and upon chronic expression of metastable proteins. We propose that transcellular chaperone signaling provides a critical control step for the PN to maintain cellular and organismal health span. PMID:25030693

  2. NAP1 family histone chaperones are required for somatic homologous recombination in Arabidopsis.

    PubMed

    Gao, Juan; Zhu, Yan; Zhou, Wangbin; Molinier, Jean; Dong, Aiwu; Shen, Wen-Hui

    2012-04-01

    Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR. PMID:22534127

  3. CrAgDb--a database of annotated chaperone repertoire in archaeal genomes.

    PubMed

    Rani, Shikha; Srivastava, Abhishikha; Kumar, Manish; Goel, Manisha

    2016-03-01

    Chaperones are a diverse class of ubiquitous proteins that assist other cellular proteins in folding correctly and maintaining their native structure. Many different chaperones cooperate to constitute the 'proteostasis' machinery in the cells. It has been proposed earlier that archaeal organisms could be ideal model systems for deciphering the basic functioning of the 'protein folding machinery' in higher eukaryotes. Several chaperone families have been characterized in archaea over the years but mostly one protein at a time, making it difficult to decipher the composition and mechanistics of the protein folding system as a whole. In order to deal with these lacunae, we have developed a database of all archaeal chaperone proteins, CrAgDb (Chaperone repertoire in Archaeal genomes). The data have been presented in a systematic way with intuitive browse and search facilities for easy retrieval of information. Access to these curated datasets should expedite large-scale analysis of archaeal chaperone networks and significantly advance our understanding of operation and regulation of the protein folding machinery in archaea. Researchers could then translate this knowledge to comprehend the more complex protein folding pathways in eukaryotic systems. The database is freely available at http://14.139.227.92/mkumar/cragdb/. PMID:26862144

  4. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

    PubMed

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-07-17

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. PMID:25036637

  5. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    PubMed Central

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-01-01

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins. PMID:26580605

  6. Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis.

    PubMed

    Shonhai, Addmore; Maier, Alexander G; Przyborski, Jude M; Blatch, Gregory L

    2011-02-01

    Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40. PMID:20955165

  7. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals

    SciTech Connect

    Sasaki, Kentaro; Kim, Myung-Hee; Imai, Ryozo

    2007-12-21

    Bacterial cold shock proteins (CSPs) are RNA chaperones that unwind RNA secondary structures. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 (AtCSP2) contains a domain that is shared with bacterial CSPs. Here we showed that AtCSP2 binds to RNA and unwinds nucleic acid duplex. Heterologous expression of AtCSP2 complemented cold sensitivity of an Escherichia coli csp quadruple mutant, indicating that AtCSP2 function as a RNA chaperone in E. coli. AtCSP2 mRNA and protein levels increased during cold acclimation, but the protein accumulation was most prominent after 10 days of cold treatment. AtCSP2 promoter::GUS transgenic plants revealed that AtCSP2 is expressed only in root and shoot apical regions during vegetative growth but is expressed in reproductive organs such as pollens, ovules and embryos. These data indicated that AtCSP2 is involved in developmental processes as well as cold adaptation. Localization of AtCSP2::GFP in nucleolus and cytoplasm suggested different nuclear and cytosolic RNA targets.

  8. Chemical chaperones reduce ER stress and adipose tissue inflammation in high fat diet-induced mouse model of obesity

    PubMed Central

    Chen, Yaqin; Wu, Zhihong; Zhao, Shuiping; Xiang, Rong

    2016-01-01

    Obesity, which is characteristic by chronic inflammation, is defined as abnormal or excessive fat accumulation in adipose tissues. Endoplasmic reticulum (ER) stress is increased in adipose tissue of obese state and is known to be strongly associated with chronic inflammation. The aim of this study was to investigate the effect of ER stress on adipokine secretion in obese mice and explore the potential mechanisms. In this study, we found high-fat diet induced-obesity contributed to strengthened ER stress and triggered chronic inflammation in adipose tissue. Chemical chaperones, 4-PBA and TUDCA, modified metabolic disorders and decreased the levels of inflammatory cytokines in obese mice fed a high-fat diet. The alleviation of ER stress is in accordance with the decrease of free cholesterol in adipose tissue. Furthermore chemical chaperones suppress NF-κB activity in adipose tissue of obese mice in vivo. In vitro studies showed IKK/NF-κB may be involved in the signal transduction of adipokine secretion dysfunction induced by ER stress. The present study revealed the possibility that inhibition of ER stress may be a novel drug target for metabolic abnormalities associated with obesity. Further studies are now needed to characterize the initial incentive of sustained ER stress in obese. PMID:27271106

  9. Chemical chaperones reduce ER stress and adipose tissue inflammation in high fat diet-induced mouse model of obesity.

    PubMed

    Chen, Yaqin; Wu, Zhihong; Zhao, Shuiping; Xiang, Rong

    2016-01-01

    Obesity, which is characteristic by chronic inflammation, is defined as abnormal or excessive fat accumulation in adipose tissues. Endoplasmic reticulum (ER) stress is increased in adipose tissue of obese state and is known to be strongly associated with chronic inflammation. The aim of this study was to investigate the effect of ER stress on adipokine secretion in obese mice and explore the potential mechanisms. In this study, we found high-fat diet induced-obesity contributed to strengthened ER stress and triggered chronic inflammation in adipose tissue. Chemical chaperones, 4-PBA and TUDCA, modified metabolic disorders and decreased the levels of inflammatory cytokines in obese mice fed a high-fat diet. The alleviation of ER stress is in accordance with the decrease of free cholesterol in adipose tissue. Furthermore chemical chaperones suppress NF-κB activity in adipose tissue of obese mice in vivo. In vitro studies showed IKK/NF-κB may be involved in the signal transduction of adipokine secretion dysfunction induced by ER stress. The present study revealed the possibility that inhibition of ER stress may be a novel drug target for metabolic abnormalities associated with obesity. Further studies are now needed to characterize the initial incentive of sustained ER stress in obese. PMID:27271106

  10. Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris.

    PubMed

    Yang, Jun; Lu, Zhipeng; Chen, Jiawei; Chu, Pinpin; Cheng, Qingmei; Liu, Jie; Ming, Feiping; Huang, Chaoyuan; Xiao, Anji; Cai, Haiming; Zhang, Linghua

    2016-06-01

    Mammalian peptidoglycan recognition proteins (PGLYRPs) are highly conserved pattern-recognition molecules of the innate immune system with considerable bactericidal activity, which manifest their potential values for the application to food and pharmaceutical industry. However, the effective expression of porcine PGLYRP-1 in Pichia pastoris has not been reported so far. In this study, expression in P. pastoris was explored as an efficient way to produce functional porcine PGLYRP-1. Cooperation of chaperones co-expression and gene dosage (including protein disulfide isomerase (PDI)/binding protein (BiP) and pglyrp-1) were used to enhance functional expression of antimicrobial protein in P. pastoris. Overexpression of PDI was certainly able to increase secretion level of PGLYRP-1 protein because the increase in secreted PGLYRP-1 secretion was correlated with the copy numbers of PDI in high copy pglyrp-1 clones. However, co-expression of BiP was proved to be detrimental to PGLYRP-1 secretion. In addition, we also found that excessive expression of PDI and/or BiP could decrease the mRNA expression of pglyrp-1 gene. This showed that PDI and BiP as the target genes of unfolded protein response (UPR) might regulate the transcription of the target protein. These data demonstrated for the first time that the combination of chaperones and gene dosages could improve the yield of PGLYRP-1, which could facilitate the application to food and pharmaceutical industry. PMID:26883349

  11. Dictyostelium discoideum Ax2 as an Assay System for Screening of Pharmacological Chaperones for Phenylketonuria Mutations.

    PubMed

    Kim, Yu-Min; Yang, Yun Gyeong; Kim, Hye-Lim; Park, Young Shik

    2015-06-01

    In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations. PMID:25563416

  12. Possible involvement of the Sigma-1 receptor chaperone in chemotherapeutic-induced neuropathic pain.

    PubMed

    Tomohisa, Mori; Junpei, Ohya; Aki, Masumoto; Masato, Harumiya; Mika, Fukase; Kazumi, Yoshizawa; Teruo, Hayashi; Tsutomu, Suzuki

    2015-11-01

    Previous studies have shown that ligands of the sigma-1 receptor chaperone (Sig-1R) regulate pain-related behaviors. Clinical use of chemotherapeutics is often compromised due to their adverse side effects, particularly those related to neuropathy. Previous studies have shown that repeated administration of oxaliplatin and paclitaxel produces neuropathy in rodents. Therefore, the aim of the present study was to clarify the involvement of the Sig-1R in chemotherapeutic-induced neuropathy by examining the effects of oxaliplatin and paclitaxel on the Sig-1R levels in the spinal cord, and by examining the effects of Sig-1R agonist and antagonist on oxaliplatin- and paclitaxel-induced neuropathy in rats. Chemotherapeutic-induced neuropathic pain was accompanied by a significant reduction of the Sig-1R level in the spinal cord. Furthermore, the administration of paclitaxel to CHO cells that stably overexpressed Sig-1Rs induced the clustering of Sig-1Rs. We also found that the Sig-1R agonist SA4503 potently inhibited the neuropathy induced by oxaliplatin- and paclitaxel, whereas this action was abolished by the Sig-1R antagonist NE-100. These results suggest that the reduction of Sig-1R activity is involved in chemotherapeutic-induced neuropathy, and the Sig-1R agonist SA4503 could serve as a potential candidate for the treatment of chemotherapeutic-induced neuropathy. PMID:26234785

  13. Coordination of chemical (trimethylamine oxide) and molecular (heat shock protein 70) chaperone responses to heat stress in elasmobranch red blood cells.

    PubMed

    Kolhatkar, Ashra; Robertson, Cayleih E; Thistle, Maria E; Gamperl, A Kurt; Currie, Suzanne

    2014-01-01

    Chemical and molecular chaperones are organic compounds that protect and stabilize proteins from damage and aggregation as a result of cellular stress. Using the dogfish (Squalus acanthias) red blood cell (RBC) as a model, we examined whether elasmobranch cells with naturally high concentrations of the chemical chaperone trimethylamine oxide (TMAO) would induce the molecular chaperone heat shock protein 70 (HSP70) when exposed to an acute thermal stress. Our hypothesis was that TMAO is itself capable of preventing damage and preserving cellular function during thermal stress and thus that the heat shock response would be inhibited/diminished. We incubated RBCs in vitro with and without physiologically relevant concentrations of TMAO at 13°C and then exposed cells to a 1-h acute heat shock at 24°C. HSP70 protein expression was elevated in dogfish RBCs after the acute heat stress, but this induction was inhibited by extracellular TMAO. Regardless of the presence of TMAO and/or HSP70, we did not observe any cell damage, as indicated by changes in caspase 3/7 activity, protein carbonyls, membrane viability, or levels of ubiquitin. We also saw no change in RBC cell function, as determined by hemoglobin oxygen affinity or carrying capacity, in cells lacking the heat shock response but protected by TMAO. This study demonstrates that there is cellular coordination between chemical and molecular chaperones in response to an acute thermal stress in dogfish RBCs and suggests that TMAO has a thermoprotective role in these cells, thus eliminating the need for a heat shock response. PMID:25244377

  14. Proteins with RNA Chaperone Activity: A World of Diverse Proteins with a Common Task—Impediment of RNA Misfolding

    PubMed Central

    Semrad, Katharina

    2011-01-01

    Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied in vitro and in vivo using oligonucleotide- or ribozyme-based assays. Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here. PMID:21234377

  15. Sulphur shuttling across a chaperone during molybdenum cofactor maturation

    NASA Astrophysics Data System (ADS)

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I.; Toci, René; Mendel, Ralf R.; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-01

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP—used as a surrogate of the molybdenum cofactor’s nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  16. Protein chaperones: a composition of matter review (2008 – 2013)

    PubMed Central

    Taldone, Tony; Patel, Hardik J; Bolaender, Alexander; Patel, Maulik R; Chiosis, Gabriela

    2014-01-01

    Introduction Heat shock proteins (Hsps) are proteins with important functions in regulating disease phenotypes. Historically, Hsp90 has first received recognition as a target in cancer, with consequent efforts extending its potential role to other diseases. Hsp70 has also attracted interest as a therapeutic target for its role as a co-chaperone to Hsp90 as well as its own anti-apoptotic roles. Areas covered Herein, patents from 2008 to 2013 are reviewed to identify those that disclose composition of matter claimed to inhibit Hsp90 or Hsp70. Expert opinion For Hsp90, there has been considerable creativity in the discovery of novel pharmacophores that fall outside the three initially discovered scaffolds (i.e., ansamycins, resorcinols and purines). Nonetheless, much of the patent literature appears to build on previously reported structure activity relationship through slight modifications of Hsp90 inhibitor space by finding weaknesses in existing patents. The major goal of future development of Hsp90 inhibitors is not necessarily identifying better molecules but rather understanding how to rationally use these agents in the clinic. The development of Hsp70 inhibitors has lagged behind. It will require a more concerted effort from the drug discovery community in order to begin to realize the potential of this target. PMID:24742089

  17. Chaperone-Mediated Autophagy and Mitochondrial Homeostasis in Parkinson's Disease

    PubMed Central

    Gao, Guodong; Mao, Zixu; Yang, Qian

    2016-01-01

    Parkinson's disease (PD), a complex neurodegenerative disorder, is pathologically characterized by the formation of Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial dysfunction is considered to be one of the most important causative mechanisms. In addition, dysfunction of chaperone-mediated autophagy (CMA), one of the lysosomal proteolytic pathways, has been shown to play an important role in the pathogenesis of PD. An exciting and important development is recent finding that CMA and mitochondrial quality control may be linked. This review summarizes the studies revealing the link between autophagy and mitochondrial function. Discussions are focused on the connections between CMA and mitochondrial failure and on the role of MEF2D, a neuronal survival factor, in mediating the regulation of mitochondria in the context of CMA. These new findings highlight the need to further explore the possibility of targeting the MEF2D-mitochondria-CMA network in both understanding the PD pathogenesis and developing novel therapeutic strategies. PMID:27413575

  18. Prion-impairing mutations in Hsp70 chaperone Ssa1

    PubMed Central

    Needham, Patrick G.; Masison, Daniel C.

    2008-01-01

    We previously described many Hsp70 Ssa1p mutants that impair [PSI+] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1L483W ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones. Ssa1A17V and Ssa1R34K ATPase activities were nearly wild type but both showed increased stimulation by substrates. Peptide binding and reactivation of denatured luciferase were enhanced in Ssa1A17V and Ssa1R34K but compromised in Ssa1L483W. The nucleotide exchange factor Fes1 influenced ATPase of wild type Ssa1 and each mutant differently. Partial protease digestion uncovered similar and distinct conformational changes of the substrate-binding domain among the three mutants. Our data suggest that prion-impairing mutations of Ssa1 can increase or decrease substrate interactions, alter the Hsp70 reaction cycle at different points and impair normal NBD-SBD cooperation. PMID:18706386

  19. Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

    PubMed

    Chan, Sze Wah Samuel; Yau, Jason; Ing, Christopher; Liu, Kaiyin; Farber, Patrick; Won, Amy; Bhandari, Vaibhav; Kara-Yacoubian, Nareg; Seraphim, Thiago V; Chakrabarti, Nilmadhab; Kay, Lewis E; Yip, Christopher M; Pomès, Régis; Sharpe, Simon; Houry, Walid A

    2016-07-01

    Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation. PMID:27265850

  20. Withaferin A Analogs That Target the AAA+ Chaperone p97.

    PubMed

    Tao, Shasha; Tillotson, Joseph; Wijeratne, E M Kithsiri; Xu, Ya-ming; Kang, MinJin; Wu, Tongde; Lau, Eric C; Mesa, Celestina; Mason, Damian J; Brown, Robert V; La Clair, James J; Gunatilaka, A A Leslie; Zhang, Donna D; Chapman, Eli

    2015-08-21

    Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both in vitro and in cell models in addition to inhibiting the proteasome in vitro. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures' intriguing molecular puzzles. PMID:26006219

  1. Diabetic Peripheral Neuropathy: Should a Chaperone Accompany Our Therapeutic Approach?

    PubMed Central

    Farmer, Kevin L.; Li, Chengyuan

    2012-01-01

    Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that is associated with axonal atrophy, demyelination, blunted regenerative potential, and loss of peripheral nerve fibers. The development and progression of DPN is due in large part to hyperglycemia but is also affected by insulin deficiency and dyslipidemia. Although numerous biochemical mechanisms contribute to DPN, increased oxidative/nitrosative stress and mitochondrial dysfunction seem intimately associated with nerve dysfunction and diminished regenerative capacity. Despite advances in understanding the etiology of DPN, few approved therapies exist for the pharmacological management of painful or insensate DPN. Therefore, identifying novel therapeutic strategies remains paramount. Because DPN does not develop with either temporal or biochemical uniformity, its therapeutic management may benefit from a multifaceted approach that inhibits pathogenic mechanisms, manages inflammation, and increases cytoprotective responses. Finally, exercise has long been recognized as a part of the therapeutic management of diabetes, and exercise can delay and/or prevent the development of painful DPN. This review presents an overview of existing therapies that target both causal and symptomatic features of DPN and discusses the role of up-regulating cytoprotective pathways via modulating molecular chaperones. Overall, it may be unrealistic to expect that a single pharmacologic entity will suffice to ameliorate the multiple symptoms of human DPN. Thus, combinatorial therapies that target causal mechanisms and enhance endogenous reparative capacity may enhance nerve function and improve regeneration in DPN if they converge to decrease oxidative stress, improve mitochondrial bioenergetics, and increase response to trophic factors. PMID:22885705

  2. Stress chaperone GRP-78 functions in mineralized matrix formation.

    PubMed

    Ravindran, Sriram; Gao, Qi; Ramachandran, Amsaveni; Blond, Sylvie; Predescu, Sanda A; George, Anne

    2011-03-18

    Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78. PMID:21239500

  3. Molecular chaperones and heat shock proteins in atherosclerosis

    PubMed Central

    Xu, Qingbo; Metzler, Bernhard; Jahangiri, Marjan

    2012-01-01

    In response to stress stimuli, mammalian cells activate an ancient signaling pathway leading to the transient expression of heat shock proteins (HSPs). HSPs are a family of proteins serving as molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. Physiologically, HSPs play a protective role in the homeostasis of the vessel wall but have an impact on immunoinflammatory processes in pathological conditions involved in the development of atherosclerosis. For instance, some members of HSPs have been shown to have immunoregulatory properties and modification of innate and adaptive response to HSPs, and can protect the vessel wall from the disease. On the other hand, a high degree of sequence homology between microbial and mammalian HSPs, due to evolutionary conservation, carries a risk of misdirected autoimmunity against HSPs expressed on the stressed cells of vascular endothelium. Furthermore, HSPs and anti-HSP antibodies have been shown to elicit production of proinflammatory cytokines. Potential therapeutic use of HSP in prevention of atherosclerosis involves achieving optimal balance between protective and immunogenic effects of HSPs and in the progress of research on vaccination. In this review, we update the progress of studies on HSPs and the integrity of the vessel wall, discuss the mechanism by which HSPs exert their role in the disease development, and highlight the potential clinic translation in the research field. PMID:22058161

  4. Pharmacological Chaperones for Misfolded Gonadotropin-Releasing Hormone Receptors

    PubMed Central

    Conn, P. Michael; Ulloa-Aguirre, Alfredo

    2011-01-01

    Structural alterations provoked by mutations or genetic variations in the gene sequence of G protein-coupled receptors may lead to abnormal function of the receptor molecule and, ultimately, to disease. While some mutations lead to changes in domains involved in agonist binding, receptor activation or coupling to effectors, others may cause misfolding and lead to retention/degradation of the protein molecule by the quality control system of the cell. Several strategies, including genetic, chemical and pharmacological approaches have been shown to rescue function of trafficking-defective misfolded G protein-coupled receptors. Among these, pharmacological strategies offer the most promising therapeutic tool to promote proper trafficking of misfolded proteins to the plasma membrane. Pharmacological chaperones or “pharmacoperones,” are small compounds that permeate the plasma membrane, enter cells, and bind selectively to misfolded proteins and correct folding allowing routing of the target protein to the plasma membrane, where the receptor may bind and respond to agonist stimulation. In this review we describe new therapeutic opportunities based on misfolding of otherwise functional human gonadotropin-releasing hormone receptors. This particular receptor is highly sensitive to single changes in chemical charge and its intracellular traffic is delicately balanced between expression at the plasma membrane or retention/degradation in the endoplasmic reticulum; it is, therefore, a particularly instructive model to understand both protein routing and the molecular mechanisms whereby pharmacoperones rescue misfolded intermediates or conformationally defective receptors. PMID:21907908

  5. Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation*

    PubMed Central

    Ravindran, Sriram; Gao, Qi; Ramachandran, Amsaveni; Blond, Sylvie; Predescu, Sanda A.; George, Anne

    2011-01-01

    Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78. PMID:21239500

  6. High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones.

    PubMed

    Tropak, Michael B; Blanchard, Jan E; Withers, Stephen G; Brown, Eric D; Mahuran, Don

    2007-02-01

    The adult forms of Tay-Sachs and Sandhoff diseases result when the activity of beta-hexosaminidase A (Hex) falls below approximately 10% of normal due to decreased transport of the destabilized mutant enzyme to the lysosome. Carbohydrate-based competitive inhibitors of Hex act as pharmacological chaperones (PC) in patient cells, facilitating exit of the enzyme from the endoplasmic reticulum, thereby increasing the mutant Hex protein and activity levels in the lysosome 3- to 6-fold. To identify drug-like PC candidates, we developed a fluorescence-based real-time enzyme assay and screened the Maybridge library of 50,000 compounds for inhibitors of purified Hex. Three structurally distinct micromolar competitive inhibitors, a bisnaphthalimide, nitro-indan-1-one, and pyrrolo[3,4-d]pyridazin-1-one were identified that specifically increased lysosomal Hex protein and activity levels in patient fibroblasts. These results validate screening for inhibitory compounds as an approach to identifying PCs. PMID:17317569

  7. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    PubMed

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc. PMID:27187154

  8. Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

    PubMed Central

    Finka, Andrija; Mattoo, Rayees U. H.

    2010-01-01

    Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844

  9. NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape

    PubMed Central

    Holmberg Olausson, Karl; Elsir, Tamador; Moazemi Goudarzi, Kaveh; Nistér, Monica; Lindström, Mikael S.

    2015-01-01

    Glioblastoma (grade IV glioma) is the most common and aggressive adult brain tumor. A better understanding of the biology of glioblastoma cells is crucial to identify molecular targets stimulating cell death. NPM1 (nucleophosmin) is a multifunctional chaperone that plays an important role in cancer development. Herein, NPM1 was analyzed by immunohistochemistry in human astrocytic gliomas. NPM1 was detected in all tumors but with a significantly higher staining intensity in grade IV than in low grade tumors. Depletion of NPM1 had only modest effects on the viability of U251MG, U1242MG, and U343MGa Cl2:6 glioma cells, despite alterations in nucleolar morphology. Glioma cell cultures depleted of NPM1 exposed to micromolar levels of actinomycin D were more prone to cell death (apoptosis) compared to cultures retaining NPM1. We had previously found that NPM1 binds to linker histone H1.5. Here we could show that silencing of H1.5 triggered glioma cell apoptosis as evidenced by a marked increase in both the numbers of cleaved caspase-3+ cells and in the amounts of cleaved PARP. Enforced expression of NPM1 suppressed apoptosis in H1.5 depleted glioma cells. Although our studies would suggest little effectiveness of targeting NPM1 alone there could be potential using it as a combination treatment. PMID:26559910

  10. An essential role for chaperone-mediated autophagy in cell cycle progression

    PubMed Central

    Hubbi, Maimon E; Semenza, Gregg L

    2015-01-01

    Hypoxia has long been known to serve as a stimulus for cell cycle arrest. Hypoxia-mediated cell cycle arrest is mediated through the actions of HIF1α (hypoxia inducible factor 1, α subunit [basic helix-loop-helix transcription factor]), which has a nontranscriptional role as an inhibitor of MCM (minichromosome maintenance complex component) helicase activity. We identified chaperone-mediated autophagy as a pathway for selective degradation of HIF1α through lysosomes prior to the onset of DNA replication. CDK2 (cyclin-dependent kinase 2) mediates degradation of HIF1α at the G1/S transition, whereas CDK1 (cyclin-dependent kinase 1) increases HIF1α levels and transcriptional activity prior to the onset of G1 phase. Lysosomal inhibitors induce cell cycle arrest, which is recovered by knockdown of HIF1α and EPAS1/HIF2α. These findings establish lysosomes as essential regulators of cell cycle progression through the degradation of HIF1α. PMID:25945892

  11. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

    PubMed Central

    Nijtmans, Leo G.J.; de Jong, Liesbeth; Artal Sanz, Marta; Coates, Philip J.; Berden, Jan A.; Willem Back, Jaap; Muijsers, Anton O.; van der Spek, Hans; Grivell, Les A.

    2000-01-01

    Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins. PMID:10835343

  12. Silencing of natural transformation by an RNA chaperone and a multitarget small RNA.

    PubMed

    Attaiech, Laetitia; Boughammoura, Aïda; Brochier-Armanet, Céline; Allatif, Omran; Peillard-Fiorente, Flora; Edwards, Ross A; Omar, Ayat R; MacMillan, Andrew M; Glover, Mark; Charpentier, Xavier

    2016-08-01

    A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species. PMID:27432973

  13. Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle

    PubMed Central

    Winter, Lilli; Staszewska, Ilona; Mihailovska, Eva; Fischer, Irmgard; Goldmann, Wolfgang H.; Schröder, Rolf; Wiche, Gerhard

    2014-01-01

    The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD. PMID:24487589

  14. PYRIMETHAMINE AS A POTENTIAL PHARMACOLOGICAL CHAPERONE FOR LATE-ONSET FORMS OF GM2 GANGLIOSIDOSIS

    PubMed Central

    Maegawa, Gustavo H. B.; Tropak, Michael; Butner, Justin; Stockley, Tracy; Kok, Fernando; Clarke, Joe T. R.; Mahuran, Don J.

    2007-01-01

    Late-onset GM2-gangliosidosis (GM2) is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric β-hexosaminidase A (Hex A, αβ). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the ER. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 FDA-approved compounds identified pyrimethamine (PYR) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 α-mutations, 2 novel), and 7 Sandhoff (9 β-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, αG269S, showed significant increases in residual Hex A activity, as did all 7 of the β-mutants tested. Cells responding to PC-treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable α-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I β-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 patient cells. PMID:17237499

  15. Quercetin mediated reduction of angiogenic markers and chaperones in DLA-induced solid tumours.

    PubMed

    Anand, Kushi; Asthana, Pallavi; Kumar, Anup; Ambasta, Rashmi K; Kumar, Pravir

    2011-01-01

    Diet-derived flavonoids, in particular quercetin, may play advantageous roles by preventing or/and inhibiting oncogenesis. Evidence suggests that quercetin can elicit various properties depending on the cell type. The aim of this study was to evaluate its effects on Dalton's lymphoma ascites (DLA) induced solid tumours and to identify the target(s) of action. We addressed this question by inducing subcutaneous solid tumours in Swiss albino mice and investigated whether the quercetin affects essential biological processes that are responsible for tumour growth, morphology, angiogenesis and apoptosis. We also studied influence on several heat shock proteins (HSPs). Our findings demonstrate that intra-tumour administration of quercetin results in decreased volume/weight. Furthermore, we demonstrate that quercetin promotes apoptosis of cancer cells by down-regulating the levels of Hsp90 and Hsp70. Depletion of these two chaperones by quercetin might result in triggering of caspase-3 in treated tumours. Moreover, it also down-regulated the expression of major key angiogenic or pro-angiogenic factors, like HIF-1α and VEGF In addition, H and E staining together with immunofluorescence of fixed tumour tissue provided evidence in support of increased cell death in quercetin-treated mice. PMID:22393949

  16. Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis.

    PubMed

    Maegawa, Gustavo H B; Tropak, Michael; Buttner, Justin; Stockley, Tracy; Kok, Fernando; Clarke, Joe T R; Mahuran, Don J

    2007-03-23

    Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric beta-hexosaminidase A (Hex A, alphabeta). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 alpha-mutations, 2 novel) and 7 Sandhoff (9 beta-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, alphaG269S, showed significant increases in residual Hex A activity, as did all 7 of the beta-mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable alpha-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I beta-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells. PMID:17237499

  17. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

    PubMed Central

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-01-01

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis. PMID:27515126

  18. The Chaperone Network Connected to Human Ribosome-Associated Complex ▿ ‡ †

    PubMed Central

    Jaiswal, Himjyot; Conz, Charlotte; Otto, Hendrik; Wölfle, Tina; Fitzke, Edith; Mayer, Matthias P.; Rospert, Sabine

    2011-01-01

    Mammalian ribosome-associated complex (mRAC), consisting of the J-domain protein MPP11 and the atypical Hsp70 homolog (70-homolog) Hsp70L1, can partly complement the function of RAC, which is the homologous complex from Saccharomyces cerevisiae. RAC is the J-domain partner exclusively of the 70-homolog Ssb, which directly and independently of RAC binds to the ribosome. We here show that growth defects due to mRAC depletion in HeLa cells resemble those of yeast strains lacking RAC. Functional conservation, however, did not extend to the 70-homolog partner of mRAC. None of the major human 70-homologs was able to complement the growth defects of yeast strains lacking Ssb or was bound to ribosomes in an Ssb-like manner. Instead, our data suggest that mRAC was a specific partner of human Hsp70 but not of its close homolog Hsc70. On a mechanistic level, ATP binding, but not ATP hydrolysis, by Hsp70L1 affected mRAC's function as a J-domain partner of Hsp70. The combined data indicate that, while functionally conserved, yeast and mammalian cells have evolved distinct solutions to ensure that Hsp70-type chaperones can efficiently assist the biogenesis of newly synthesized polypeptide chains. PMID:21245388

  19. The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

    PubMed Central

    Boulon, Séverine; Marmier-Gourrier, Nathalie; Pradet-Balade, Bérengère; Wurth, Laurence; Verheggen, Céline; Jády, Beáta E.; Rothé, Benjamin; Pescia, Christina; Robert, Marie-Cécile; Kiss, Tamás; Bardoni, Barbara; Krol, Alain; Branlant, Christiane; Allmang, Christine; Bertrand, Edouard; Charpentier, Bruno

    2008-01-01

    RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth. PMID:18268104

  20. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    NASA Astrophysics Data System (ADS)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  1. Functions of the histone chaperone nucleolin in diseases.

    PubMed

    Storck, Sébastien; Shukla, Manu; Dimitrov, Stefan; Bouvet, Philippe

    2007-01-01

    Alteration of nuclear morphology is often used by pathologist as diagnostic marker for malignancies like cancer. In particular, the staining of cells by the silver staining methods (AgNOR) has been proved to be an important tool for predicting the clinical outcome of some cancer diseases. Two major argyrophilic proteins responsible for the strong staining of cells in interphase are the nucleophosmin (B23) and the nucleolin (C23) nucleolar proteins. Interestingly these two proteins have been described as chromatin associated proteins with histone chaperone activities and also as proteins able to regulate chromatin transcription. Nucleolin seems to be over-expressed in highly proliferative cells and is involved in many aspect of gene expression: chromatin remodeling, DNA recombination and replication, RNA transcription by RNA polymerase I and II, rRNA processing, mRNA stabilisation, cytokinesis and apoptosis. Interestingly, nucleolin is also found on the cell surface in a wide range of cancer cells, a property which is being used as a marker for the diagnosis of cancer and for the development of anti-cancer drugs to inhibit proliferation of cancer cells. In addition to its implication in cancer, nucleolin has been described not only as a marker or as a protein being involved in many diseases like viral infections, autoimmune diseases, Alzheimer's disease pathology but also in drug resistance. In this review we will focus on the chromatin associated functions of nucleolin and discuss the functions of nucleolin or its use as diagnostic marker and as a target for therapy PMID:17484127

  2. Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis.

    PubMed

    Chow, Ari M; Steel, Rohan; Anderson, Robin L

    2009-05-01

    In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity. PMID:18819021

  3. Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

    PubMed

    Ecroyd, Heath; Meehan, Sarah; Horwitz, Joseph; Aquilina, J Andrew; Benesch, Justin L P; Robinson, Carol V; Macphee, Cait E; Carver, John A

    2007-01-01

    AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases. PMID:16928191

  4. ATP-dependent molecular chaperones in plastids--More complex than expected.

    PubMed

    Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

    2015-09-01

    Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25596449

  5. Targeting Hsp90 and its co-chaperones to treat Alzheimer’s disease

    PubMed Central

    Blair, Laura J.; Sabbagh, Jonathan J.; Dickey, Chad A.

    2015-01-01

    Introduction Alzheimer’s disease (AD), characterized by the accumulation of hyperphosphorylated tau and beta amyloid (Aβ), currently lacks effective treatment. Chaperone proteins, such as the heat shock protein (Hsp) 90, form macromolecular complexes with co-chaperones, which can regulate tau metabolism and Aβ processing. While small molecule inhibitors of Hsp90 have been successful at ameliorating tau and Aβ burden, their development into drugs to treat disease has been slow due to the off- and on-target effects of this approach as well as challenges with the pharmacology of current scaffolds. Thus, other approaches are being developed to improve these compounds and to target co-chaperones of Hsp90 in an effort to limit these liabilities. Areas Covered This article discusses the most current developments in Hsp90 inhibitors including advances in blood-brain barrier permeability, decreased toxicity, and homolog-specific small molecule inhibitors. In addition, we discuss current strategies targeting Hsp90 co-chaperones rather than Hsp90 itself to reduce off-target effects. Expert Opinion While Hsp90 inhibitors have proven their efficacy at reducing tau pathology, they have yet to meet with success in the clinic. The development of Hsp90/tau complex specific inhibitors and further development of Hsp90 co-chaperone specific drugs should yield more potent, less toxic therapeutics. PMID:25069659

  6. Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

    PubMed Central

    Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; Argüello, José M.; Merchant, Sabeeha S.

    2014-01-01

    Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. We also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution. PMID:25468978

  7. HtrA chaperone activity contributes to host cell binding in Campylobacter jejuni

    PubMed Central

    2011-01-01

    Background Acute gastroenteritis caused by the food-borne pathogen Campylobacter jejuni is associated with attachment of bacteria to the intestinal epithelium and subsequent invasion of epithelial cells. In C. jejuni, the periplasmic protein HtrA is required for efficient binding to epithelial cells. HtrA has both protease and chaperone activity, and is important for virulence of several bacterial pathogens. Results The aim of this study was to determine the role of the dual activities of HtrA in host cell interaction of C. jejuni by comparing an htrA mutant lacking protease activity, but retaining chaperone activity, with a ΔhtrA mutant and the wild type strain. Binding of C. jejuni to both epithelial cells and macrophages was facilitated mainly by HtrA chaperone activity that may be involved in folding of outer membrane adhesins. In contrast, HtrA protease activity played only a minor role in interaction with host cells. Conclusion We show that HtrA protease and chaperone activities contribute differently to C. jejuni's interaction with mammalian host cells, with the chaperone activity playing the major role in host cell binding. PMID:21939552

  8. Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

    PubMed Central

    Kuehnert, Julia; Sommer, Gunhild; Zierk, Avery W.; Fedarovich, Alena; Brock, Alexander; Fedarovich, Dzmitry; Heise, Tilman

    2015-01-01

    The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation. PMID:25520193

  9. The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins

    PubMed Central

    Singhal, Kushagra; Vreede, Jocelyne; Mashaghi, Alireza; Tans, Sander J.; Bolhuis, Peter G.

    2015-01-01

    How chaperones interact with protein chains to assist in their folding is a central open question in biology. Obtaining atomistic insight is challenging in particular, given the transient nature of the chaperone-substrate complexes and the large system sizes. Recent single-molecule experiments have shown that the chaperone Trigger Factor (TF) not only binds unfolded protein chains, but can also guide protein chains to their native state by interacting with partially folded structures. Here, we used all-atom MD simulations to provide atomistic insights into how Trigger Factor achieves this chaperone function. Our results indicate a crucial role for the tips of the finger-like appendages of TF in the early interactions with both unfolded chains and partially folded structures. Unfolded chains are kinetically trapped when bound to TF, which suppresses the formation of transient, non-native end-to-end contacts. Mechanical flexibility allows TF to hold partially folded structures with two tips (in a pinching configuration), and to stabilize them by wrapping around its appendages. This encapsulation mechanism is distinct from that of chaperones such as GroEL, and allows folded structures of diverse size and composition to be protected from aggregation and misfolding interactions. The results suggest that an ATP cycle is not required to enable both encapsulation and liberation. PMID:26512985

  10. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

    PubMed Central

    Mack, Korrie L.; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  11. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity.

    PubMed

    Mack, Korrie L; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  12. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  13. Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

    PubMed Central

    White, Andrew D.; Huang, Wenjun; Jiang, Shaoyi

    2012-01-01

    Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein. PMID:23260050

  14. Yeast prions are useful for studying protein chaperones and protein quality control

    PubMed Central

    Masison, Daniel C; Reidy, Michael

    2015-01-01

    Abstract Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions. PMID:26110609

  15. Hsp40 Couples with the CSPα Chaperone Complex upon Induction of the Heat Shock Response

    PubMed Central

    Gibbs, Sarah J.; Barren, Brandy; Beck, Katy E.; Proft, Juliane; Zhao, Xiaoxi; Noskova, Tatiana; Braun, Andrew P.; Artemyev, Nikolai O.; Braun, Janice E. A.

    2009-01-01

    In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPα). CSPα is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich “string” region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPα chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Gαs. In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPα complex. Association of Hsp40 with CSPα decreases CSPα-CSPα dimerization and enhances the CSPα-induced increase in steady state GTP hydrolysis of Gαs. This newly identified CSPα-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPα in neuroprotection. PMID:19242542

  16. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

    PubMed

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  17. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. PMID:26644295

  18. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility

    PubMed Central

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A–H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A–H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A–H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  19. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    PubMed Central

    Requena, Jose M.; Montalvo, Ana M.; Fraga, Jorge

    2015-01-01

    Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis. PMID:26167482

  20. Regulation of the expression of chaperone gp96 in macrophages and dendritic cells.

    PubMed

    Wolfram, Lutz; Fischbeck, Anne; Frey-Wagner, Isabelle; Wojtal, Kacper A; Lang, Silvia; Fried, Michael; Vavricka, Stephan R; Hausmann, Martin; Rogler, Gerhard

    2013-01-01

    The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD). PMID:24146856

  1. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    SciTech Connect

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin; Ahn, Sung-Min; Jang, Ho Hee; Lee, Sang Yeol

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  2. Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones

    PubMed Central

    Malinverni, Duccio; Marsili, Simone; Barducci, Alessandro; De Los Rios, Paolo

    2015-01-01

    Hsp70s are a class of ubiquitous and highly conserved molecular chaperones playing a central role in the regulation of proteostasis in the cell. Hsp70s assist a myriad of cellular processes by binding unfolded or misfolded substrates during a complex biochemical cycle involving large-scale structural rearrangements. Here we show that an analysis of coevolution at the residue level fully captures the characteristic large-scale conformational transitions of this protein family, and predicts an evolutionary conserved–and thus functional–homo-dimeric arrangement. Furthermore, we highlight that the features encoding the Hsp70 dimer are more conserved in bacterial than in eukaryotic sequences, suggesting that the known Hsp70/Hsp110 hetero-dimer is a eukaryotic specialization built on a pre-existing template. PMID:26046683

  3. A Novel Mechanism for Small Heat Shock Proteins to Function as Molecular Chaperones

    PubMed Central

    Zhang, Kaiming; Ezemaduka, Anastasia N.; Wang, Zhao; Hu, Hongli; Shi, Xiaodong; Liu, Chuang; Lu, Xinping; Fu, Xinmiao; Chang, Zengyi; Yin, Chang-Cheng

    2015-01-01

    Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and single particle 3D reconstruction that, at the low temperatures (4–25°C), CeHSP17 (a sHSP from Caenorhabditis elegans) exists as a 24-subunit spherical oligomer with tetrahedral symmetry. Our studies demonstrate that CeHSP17 forms large sheet-like super-molecular assemblies (SMAs) at the high temperatures (45–60°C), and such SMAs are apparently the form that exhibits chaperone-like activity. Our findings suggest a novel molecular mechanism for sHSPs to function as molecular chaperones. PMID:25744691

  4. Structural Basis for Protein anti-Aggregation Activity of the Trigger Factor Chaperone*

    PubMed Central

    Saio, Tomohide; Guan, Xiao; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2014-01-01

    Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins. PMID:24812405

  5. Structural basis for proteasome formation controlled by an assembly chaperone nas2.

    PubMed

    Satoh, Tadashi; Saeki, Yasushi; Hiromoto, Takeshi; Wang, Ying-Hui; Uekusa, Yoshinori; Yagi, Hirokazu; Yoshihara, Hidehito; Yagi-Utsumi, Maho; Mizushima, Tsunehiro; Tanaka, Keiji; Kato, Koichi

    2014-05-01

    Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle. PMID:24685148

  6. Structural insights on two hypothetical secretion chaperones from Xanthomonas axonopodis pv. citri.

    PubMed

    Fattori, Juliana; Prando, Alessandra; Assis, Leandro H P; Aparicio, Ricardo; Tasic, Ljubica

    2011-06-01

    Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification. PMID:21626158

  7. CHIP: a quality-control E3 ligase collaborating with molecular chaperones.

    PubMed

    Murata, Shigeo; Chiba, Tomoki; Tanaka, Keiji

    2003-05-01

    It is notable that both the chaperone and ubiquitin-proteasome systems are required for removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. Carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, has both a tetratricopeptide repeat (TPR) motif and a U-box domain. The TPR motif associates with Hsc70 and Hsp90, while the U-box domain executes a ubiquitin ligase activity. Thus, CHIP is an ideal molecule acting as a protein quality-control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. Accumulating evidence from in vitro studies indicates that this is apparently the case. Here, we present and discuss several unresolved but critical issues related to the molecular mechanism and in vivo roles of CHIP. PMID:12672450

  8. Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

    PubMed

    MacLellan, Robyn J; Tunnah, Louise; Barnett, David; Wright, Patricia A; MacCormack, Tyson; Currie, Suzanne

    2015-10-01

    Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function. PMID:26050212

  9. Chaperone heat shock protein 70 in nucleus accumbens core: a novel biological target of behavioural sensitization to morphine in rats.

    PubMed

    Wang, Yan-Ting; Qin, Wang-Jun; Liu, Qing; Li, Yu-Ling; Liang, Hui; Chen, Feng; Lawrence, Andrew J; Zhang, Xiang-Lin; Liang, Jian-Hui

    2014-03-01

    Drug addiction is a major public health issue, yet the underlying adaptation of neural networks by drugs of abuse is not fully understood. We have previously linked chaperone heat shock protein 70 (Hsp70) to drug-induced adaptations. Focusing on the NAc core and shell, the present study aims to provide further findings for our understanding of the relation between behavioural sensitization to morphine and Hsp70 at transcriptional and functional levels in rats. Firstly, we delineated the characteristics of behavioural sensitization induced by a single morphine exposure (1-10 mg/kg, s.c.). Secondly, Hsp70 protein expression in the NAc core was time- and dose-relatedly induced during the development of behavioural sensitization to a single morphine exposure in rats, and Pearson analysis indicated a positive correlation between behavioural sensitization and Hsp70 expression in NAc core. Thirdly, at the transcriptional level, intra-NAc core injection of the specific heat shock factor-I (HSF-I) inhibitor N-Formyl-3,4-methylenedioxy-benzylidine-γ-butyrolactam (KNK437) suppressed Hsp70 expression and the development of behavioural sensitization, while the HSF-I specific inducer geranylgeranylacetone (GGA) promoted both of them. Interestingly, intra-NAc shell injection of KNK437 or GGA did not affect the development of behavioural sensitization. Finally, both the functional inhibition of Hsp70 ATPase activity by methylene blue (MB), and the antagonism of Hsp70 substrate binding site (SBD) activity by pifithrin-μ (PES) impaired the development of behavioural sensitization when they were microinjected into the NAc core. Taken together, the critical involvement of chaperone Hsp70 in behavioural sensitization to morphine identifies a biological target for long-lasting adaptations with relevance to addiction. PMID:24280010

  10. Mechanism of ATPase-mediated Cu+ Export and Delivery to Periplasmic Chaperones

    PubMed Central

    Padilla-Benavides, Teresita; George Thompson, Alayna M.; McEvoy, Megan M.; Argüello, José M.

    2014-01-01

    Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu2+/+. In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu+ to target transporters or metalloenzymes. Transmembrane Cu+-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu+. Cytosolic Cu+ chaperones (CopZ) interact with a structural platform in Cu+-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu+ chaperone that supplies Cu+ to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu+ transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu+-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu+ transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu+ transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu+ transfer was minimally affected. These results show how soluble chaperones obtain Cu+ from transmembrane transporters. Furthermore, by explaining the movement of Cu+ from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu+ can be precisely directed to periplasmic targets via specific transporter-chaperone interactions. PMID:24917681

  11. The human HSP70 family of chaperones: where do we stand?

    PubMed

    Radons, Jürgen

    2016-05-01

    The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as "chaperokines" or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5'-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target. PMID:26865365

  12. The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis

    PubMed Central

    Wang, Min-Cheng; Liaw, Shwu-Jen

    2014-01-01

    Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs. PMID:24454905

  13. Chaperone-like protein HYPK and its interacting partners augment autophagy.

    PubMed

    Choudhury, Kamalika Roy; Bucha, Sudha; Baksi, Shounak; Mukhopadhyay, Debashis; Bhattacharyya, Nitai P

    2016-01-01

    To decipher the function(s) of HYPK, a huntingtin (HTT)-interacting protein with chaperone-like activity, we had previously identified 36 novel interacting partners of HYPK. Another 13 proteins were known earlier to be associated with HYPK. On the basis of analysis of the interacting partners of HYPK, it has been shown that HYPK may participate in diverse cellular functions relevant to Huntington's disease. In the present study, we identified additional 5 proteins by co-immunoprecipitation and co-localization. As of now we have 54 primary interactors of HYPK. From the database we collected 1026 unique proteins (secondary interactors) interacting with these 54 primary HYPK interacting partners. We observed that 10 primary and 91 secondary interacting proteins of HYPK are associated with two types of autophagy processes. We next tested the hypothesis that the hub, HYPK, might itself be involved in autophagy. Using mouse striatal STHdh(Q7)/Hdh(Q7) cell lines, we observed that over expression of HYPK significantly increased background cellular autophagy, while knock down of endogenous HYPK decreased the autophagy level as detected by altered LC3I conversion, BECN1 expression, cleavage of GFP from LC3-GFP, ATG5-ATG12 conjugate formation and expression of transcription factors like Tfeb, Srebp2 and Zkscan3. This result shows that HYPK, possibly with its interacting partners, induces autophagy. We further observed that N-terminal mutant HTT reduced the cellular levels of LC3II and BECN1, which could be recovered significantly upon over expression of HYPK in these cells. This result further confirms that HYPK could also be involved in clearing mutant HTT aggregates by augmenting autophagy pathway. PMID:27067261

  14. Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

    PubMed Central

    Khanna, Richie; Powe, Allan C.; Boyd, Robert; Lee, Gary; Flanagan, John J.; Benjamin, Elfrida R.

    2011-01-01

    Abstract Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed. PMID:21612550

  15. Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone.

    PubMed

    Yabukarski, Filip; Leyrat, Cedric; Martinez, Nicolas; Communie, Guillaume; Ivanov, Ivan; Ribeiro, Euripedes A; Buisson, Marlyse; Gerard, Francine C; Bourhis, Jean-Marie; Jensen, Malene Ringkjøbing; Bernadó, Pau; Blackledge, Martin; Jamin, Marc

    2016-07-01

    Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs. PMID:27107640

  16. Anti-diabetic effect of 3-hydroxy-2-naphthoic acid, an endoplasmic reticulum stress-reducing chemical chaperone.

    PubMed

    Park, Sun-Mi; Choi, Jungsook; Nam, Tae-Gyu; Ku, Jin-Mo; Jeong, Kwiwan

    2016-05-15

    Lots of experimental and clinical evidences indicate that chronic exposure to saturated fatty acids and high level of glucose is implicated in insulin resistance, beta cell failure and ultimately type 2 diabetes. In this study, we set up cell-based experimental conditions to induce endoplasmic reticulum (ER) stress and insulin resistance using high concentration of palmitate (PA). Hydroxynaphthoic acids (HNAs) were formerly identified as novel chemical chaperones to resolve ER stress induced by tunicamycin. In this study, we found the compounds have the same suppressive effect on PA-induced ER stress in HepG2 cells. The representing compound, 3-HNA reduced PA-induced phosphorylation of JNK, IKKβ and IRS1 (S307) and restored insulin signaling cascade which involves insulin receptor β, IRS1 and Akt. The insulin sensitizing effect of 3-HNA was confirmed in 3T3-L1 adipocytes, where the compound augmented insulin signaling and glucose transporter 4 (GLUT4) membrane translocation. 3-HNA also protected the pancreatic beta cells from PA-induced apoptosis by reducing ER stress. Upon 3-HNA treatment to ob/ob mice at 150mg/kg/day dosage, the diabetic parameters including glucose tolerance and systemic insulin sensitivity were significantly improved. Postmortem examination showed that 3-HNA markedly reduced ER stress and insulin resistance in the liver tissues and it sensitized insulin signaling in the liver and the skeletal muscle. Our results demonstrated that 3-HNA can sensitize insulin signaling by coping with lipotoxicity-induced ER stress as a chemical chaperone and suggested it holds therapeutic potential for insulin resistance and type 2 diabetes. PMID:26983645

  17. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

    PubMed Central

    Kuznetsov, G; Bush, K T; Zhang, P L; Nigam, S K

    1996-01-01

    The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment

  18. The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

    PubMed

    Kakoschke, Tamara; Kakoschke, Sara; Magistro, Giuseppe; Schubert, Sören; Borath, Marc; Heesemann, Jürgen; Rossier, Ombeline

    2014-01-01

    To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin. PMID:24454955

  19. Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.

    PubMed

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2015-11-01

    Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of "plasma protein" complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved. PMID:25900303

  20. Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK.

    PubMed

    Arita-Morioka, Ken-ichi; Yamanaka, Kunitoshi; Mizunoe, Yoshimitsu; Ogura, Teru; Sugimoto, Shinya

    2015-01-01

    Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 μM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs. PMID:25403660

  1. Sex, Scavengers, and Chaperones: Transcriptome Secrets of Divergent Symbiodinium Thermal Tolerances

    PubMed Central

    Levin, Rachel A.; Beltran, Victor H.; Hill, Ross; Kjelleberg, Staffan; McDougald, Diane; Steinberg, Peter D.; van Oppen, Madeleine J. H.

    2016-01-01

    Corals rely on photosynthesis by their endosymbiotic dinoflagellates (Symbiodinium spp.) to form the basis of tropical coral reefs. High sea surface temperatures driven by climate change can trigger the loss of Symbiodinium from corals (coral bleaching), leading to declines in coral health. Different putative species (genetically distinct types) as well as conspecific populations of Symbiodinium can confer differing levels of thermal tolerance to their coral host, but the genes that govern dinoflagellate thermal tolerance are unknown. Here we show physiological and transcriptional responses to heat stress by a thermo-sensitive (physiologically susceptible at 32 °C) type C1 Symbiodinium population and a thermo-tolerant (physiologically healthy at 32 °C) type C1 Symbiodinium population. After nine days at 32 °C, neither population exhibited physiological stress, but both displayed up-regulation of meiosis genes by ≥ 4-fold and enrichment of meiosis functional gene groups, which promote adaptation. After 13 days at 32 °C, the thermo-sensitive population suffered a significant decrease in photosynthetic efficiency and increase in reactive oxygen species (ROS) leakage from its cells, whereas the thermo-tolerant population showed no signs of physiological stress. Correspondingly, only the thermo-tolerant population demonstrated up-regulation of a range of ROS scavenging and molecular chaperone genes by ≥ 4-fold and enrichment of ROS scavenging and protein-folding functional gene groups. The physiological and transcriptional responses of the Symbiodinium populations to heat stress directly correlate with the bleaching susceptibilities of corals that harbored these same Symbiodinium populations. Thus, our study provides novel, foundational insights into the molecular basis of dinoflagellate thermal tolerance and coral bleaching. PMID:27301593

  2. Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

    PubMed Central

    Palm, Maria E.; Weise, Christoph F.; Lundin, Christina; Wingsle, Gunnar; Nygren, Yvonne; Björn, Erik; Naredi, Peter; Wolf-Watz, Magnus; Wittung-Stafshede, Pernilla

    2011-01-01

    Cisplatin (cisPt), Pt(NH3)2Cl2, is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1’s metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance. PMID:21482801

  3. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

    PubMed

    Whitney, Spencer M; Birch, Rosemary; Kelso, Celine; Beck, Jennifer L; Kapralov, Maxim V

    2015-03-17

    Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants. PMID:25733857

  4. Pilot study using ambroxol as a pharmacological chaperone in type 1 Gaucher disease.

    PubMed

    Zimran, Ari; Altarescu, Gheona; Elstein, Deborah

    2013-02-01

    The purpose of this pilot was to assess the tolerability and efficacy of ambroxol as a pharmacological chaperone in patients with symptomatic, type 1 Gaucher disease who present with measurable disease parameters but are not receiving enzyme replacement therapy (ERT) in order to provide proof of concept and/or ascertain the suitability of ambroxol for a larger clinical trial. The Israeli Ministry of Health Form 29c was employed to prescribe ambroxol for off-label use. Twelve patients were dispensed 2 capsules of 75 mg of ambroxol daily for 6 months. There were 8 females (66.7%). Mean age at entry was 41.1 (range: 24-63) years. Mean body weight at entry was 66.4 (range: 46.5-100) kg. One patient withdrew because of a hypersensitivity reaction, one because of elective splenectomy. No patient experienced clinically relevant deterioration in disease parameters measured. One patient achieved a robust response relative to baseline: +16.2% hemoglobin; +32.9% platelets; -2.8% liver volume; and -14.4% spleen volume. Three patients, including the above one, elected to continue on ambroxol for a further 6 months: hemoglobin levels and liver volumes were relatively stable, but platelet counts further increased in the above patient (+52.6% from baseline) and spleen volumes decreased further in all three patients (-6.4%, -18.6%, and -23.4% from baseline). Thus, ambroxol may be a safe option for Gaucher disease patients with potential disease-specific efficacy and should be expanded into a clinical trial using higher doses and placebo-controlled design. PMID:23085429

  5. Novel Strategy for Biofilm Inhibition by Using Small Molecules Targeting Molecular Chaperone DnaK

    PubMed Central

    Arita-Morioka, Ken-ichi; Yamanaka, Kunitoshi; Mizunoe, Yoshimitsu; Ogura, Teru

    2014-01-01

    Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 μM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs. PMID:25403660

  6. Sex, Scavengers, and Chaperones: Transcriptome Secrets of Divergent Symbiodinium Thermal Tolerances.

    PubMed

    Levin, Rachel A; Beltran, Victor H; Hill, Ross; Kjelleberg, Staffan; McDougald, Diane; Steinberg, Peter D; van Oppen, Madeleine J H

    2016-09-01

    Corals rely on photosynthesis by their endosymbiotic dinoflagellates (Symbiodinium spp.) to form the basis of tropical coral reefs. High sea surface temperatures driven by climate change can trigger the loss of Symbiodinium from corals (coral bleaching), leading to declines in coral health. Different putative species (genetically distinct types) as well as conspecific populations of Symbiodinium can confer differing levels of thermal tolerance to their coral host, but the genes that govern dinoflagellate thermal tolerance are unknown. Here we show physiological and transcriptional responses to heat stress by a thermo-sensitive (physiologically susceptible at 32 °C) type C1 Symbiodinium population and a thermo-tolerant (physiologically healthy at 32 °C) type C1 Symbiodinium population. After nine days at 32 °C, neither population exhibited physiological stress, but both displayed up-regulation of meiosis genes by ≥ 4-fold and enrichment of meiosis functional gene groups, which promote adaptation. After 13 days at 32 °C, the thermo-sensitive population suffered a significant decrease in photosynthetic efficiency and increase in reactive oxygen species (ROS) leakage from its cells, whereas the thermo-tolerant population showed no signs of physiological stress. Correspondingly, only the thermo-tolerant population demonstrated up-regulation of a range of ROS scavenging and molecular chaperone genes by ≥ 4-fold and enrichment of ROS scavenging and protein-folding functional gene groups. The physiological and transcriptional responses of the Symbiodinium populations to heat stress directly correlate with the bleaching susceptibilities of corals that harbored these same Symbiodinium populations. Thus, our study provides novel, foundational insights into the molecular basis of dinoflagellate thermal tolerance and coral bleaching. PMID:27301593

  7. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  8. Ribonuclease A suggests how proteins self-chaperone against amyloid fiber formation

    SciTech Connect

    Teng, Poh K.; Anderson, Natalie J.; Goldschmidt, Lukasz; Sawaya, Michael R.; Sambashivan, Shilpa; Eisenberg, David

    2012-05-29

    Genomic analyses have identified segments with high fiber-forming propensity in many proteins not known to form amyloid. Proteins are often protected from entering the amyloid state by molecular chaperones that permit them to fold in isolation from identical molecules; but, how do proteins self-chaperone their folding in the absence of chaperones? Here, we explore this question with the stable protein ribonuclease A (RNase A). We previously identified fiber-forming segments of amyloid-related proteins and demonstrated that insertion of these segments into the C-terminal hinge loop of nonfiber-forming RNase A can convert RNase A into the amyloid state through three-dimensional domain-swapping, where the inserted fiber-forming segments interact to create a steric zipper spine. In this study, we convert RNase A into amyloid-like fibers by increasing the loop length and hence conformational freedom of an endogenous fiber-forming segment, SSTSAASS, in the N-terminal hinge loop. This is accomplished by sandwiching SSTSAASS between inserted Gly residues. With these inserts, SSTSAASS is now able to form the steric zipper spine, allowing RNase A to form amyloid-like fibers. We show that these fibers contain RNase A molecules retaining their enzymatic activity and therefore native-like structure. Thus, RNase A appears to prevent fiber formation by limiting the conformational freedom of this fiber-forming segment from entering a steric zipper. Our observations suggest that proteins have evolved to self-chaperone by using similar protective mechanisms.

  9. Structural variants of yeast prions show conformer-specific requirements for chaperone activity

    PubMed Central

    Stein, Kevin C.; True, Heather L.

    2016-01-01

    Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

  10. Chaperone gelator for the chiral self-assembly of all proteinogenic amino acids and their enantiomers.

    PubMed

    Chen, Jie; Wang, Tianyu; Liu, Minghua

    2016-05-01

    A concept of a chaperone gelator that can assist non-gelator molecules to form gels is proposed. Such a new gelator was developed and found to tune all the proteinogenic l-amino acids, as well as their enantiomers into supramolecular gels. PMID:27071330

  11. Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation.

    PubMed

    Winkler, Juliane; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel

    2012-08-01

    Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils. PMID:22869599

  12. Chaperone Activity of Small Heat Shock Proteins Underlies Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis*

    PubMed Central

    Kurnellas, Michael P.; Brownell, Sara E.; Su, Leon; Malkovskiy, Andrey V.; Rajadas, Jayakumar; Dolganov, Gregory; Chopra, Sidharth; Schoolnik, Gary K.; Sobel, Raymond A.; Webster, Jonathan; Ousman, Shalina S.; Becker, Rachel A.; Steinman, Lawrence; Rothbard, Jonathan B.

    2012-01-01

    To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73–92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions. PMID:22955287

  13. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    SciTech Connect

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

  14. Chaperone network composition in Solanum lycopersicum explored by transcriptome profiling and microarray meta-analysis.

    PubMed

    Fragkostefanakis, Sotirios; Simm, Stefan; Paul, Puneet; Bublak, Daniela; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-04-01

    Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato. PMID:25124075

  15. High-resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA.

    PubMed

    Quistgaard, Esben M; Nordlund, Pär; Löw, Christian

    2012-10-01

    SlpA is a 2-domain protein consisting of an FK506-binding protein (FKBP) domain that harbors the peptidyl-prolyl cis/trans-isomerase (PPIase) active site and a small insert-in-flap (IF) domain that endows the protein with chaperone activity. We have determined the structure of SlpA from Escherichia coli at 1.35-Å resolution. The overall structure is similar to other known structures of the FKBP-IF subfamily. However, by serendipity, the linker region of the purification tag binds in the chaperone binding groove of the IF domain, making this the first structure of an FKBP-IF protein in complex with a mimic of an unfolded chaperone substrate. The linker binds by β-sheet augmentation, thus completing the incomplete β barrel of the IF domain and shielding a considerable hydrophobic surface area from the solvent. Interestingly, a proline residue in trans configuration appears to be specifically recognized in a small pocket within the binding groove. Hence, the IF domain can preselect and prealign substrates with proline residues, which may explain how it enhances the catalytic efficiency and modulates the specificity of the FKBP domain in addition to its chaperone function. Based on pulldown results, we suggest that SlpA is likely to be involved in ribosome assembly. PMID:22735173

  16. DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

    PubMed

    Fontaine, Sarah N; Zheng, Dali; Sabbagh, Jonathan J; Martin, Mackenzie D; Chaput, Dale; Darling, April; Trotter, Justin H; Stothert, Andrew R; Nordhues, Bryce A; Lussier, April; Baker, Jeremy; Shelton, Lindsey; Kahn, Mahnoor; Blair, Laura J; Stevens, Stanley M; Dickey, Chad A

    2016-07-15

    It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins. PMID:27261198

  17. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  18. Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast

    PubMed Central

    Johnson, Courtney R.; Weems, Andrew D.; Brewer, Jennifer M.; Thorner, Jeremy; McMurray, Michael A.

    2015-01-01

    Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its “G interface”) cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un­expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its “unfoldase” activity, indicating a latent “holdase” activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases. PMID:25673805

  19. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics

    PubMed Central

    Parenti, Giancarlo

    2009-01-01

    Lysosomal storage diseases (LSDs) are a group of genetic disorders due to defects in any aspect of lysosomal biology. During the past two decades, different approaches have been introduced for the treatment of these conditions. Among them, enzyme replacement therapy (ERT) represented a major advance and is used successfully in the treatment of some of these disorders. However, ERT has limitations such as insufficient biodistribution of recombinant enzymes and high costs. An emerging strategy for the treatment of LSDs is pharmacological chaperone therapy (PCT), based on the use of chaperone molecules that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking. After proof-of-concept studies, PCT is now being translated into clinical applications for Fabry, Gaucher and Pompe disease. This approach, however, can only be applied to patients carrying chaperone-responsive mutations. The recent demonstration of a synergistic effect of chaperones and ERT expands the applications of PCT and prompts a re-evaluation of their therapeutic use and potential. This review discusses the strengths and drawbacks of the potential therapies available for LSDs and proposes that future research should be directed towards the development of treatment protocols based on the combination of different therapies to improve the clinical outcome of LSD patients. PMID:20049730

  20. Silkworm hemolymph down-regulates the expression of endoplasmic reticulum chaperones under radiation-irradiation.

    PubMed

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, "ischemia-responsive protein 94 kDa" (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  1. Silkworm Hemolymph Down-Regulates the Expression of Endoplasmic Reticulum Chaperones under Radiation-Irradiation

    PubMed Central

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, “ischemia-responsive protein 94 kDa” (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  2. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    DOE PAGESBeta

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

  3. The chaperone activity and toxicity of ambroxol on Gaucher cells and normal mice.

    PubMed

    Luan, Zhuo; Li, Linjing; Higaki, Katsumi; Nanba, Eiji; Suzuki, Yoshiyuki; Ohno, Kousaku

    2013-04-01

    Gaucher disease (GD), caused by a defect of acid β-glucosidase (β-Glu), is one of the most common sphingolipidoses. Recently, ambroxol, an FDA-approved drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified as a chemical chaperone for GD. In the present study, we investigated the chaperone activity and toxicity of ambroxol on both cultured GD patient cells and normal mice. We found that ambroxol treatment significantly increased N370S, F213I, N188S/G193W and R120W mutant β-Glu activities in GD fibroblasts with low cytotoxicity. Additionally, we measured the β-Glu activity in the tissues of normal mice which received water containing increasing concentrations of ambroxol ad libitum for one week. No serious adverse effect was observed during this experiment. Ambroxol significantly increased the β-Glu activity in the spleen, heart and cerebellum of the mice. This result showed its oral availability and wide distribution and chaperone activity in the tissues, including the brain, and its lack of acute toxicity. These characteristics of ambroxol would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations. PMID:22682976

  4. Bicyclic derivatives of L-idonojirimycin as pharmacological chaperones for neuronopathic forms of Gaucher disease.

    PubMed

    Alfonso, Pilar; Andreu, Vanesa; Pino-Angeles, Almudena; Moya-García, Aurelio A; García-Moreno, M Isabel; Rodríguez-Rey, José C; Sánchez-Jiménez, Francisca; Pocoví, Miguel; Ortiz Mellet, Carmen; García Fernández, Jose M; Giraldo, Pilar

    2013-05-27

    New human β-glucocerebrosidase (GCase) ligands with rigid 1,6-anhydro-β-L-idonojirimycin cores have been designed with the aid of molecular modeling. Efficient pharmacological chaperones for the L444P (trafficking-incompetent) mutant GCase enzyme associated with type 2 and 3 Gaucher disease (GD) were identified. PMID:23606264

  5. Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM1 gangliosidosis.

    PubMed

    Aguilar-Moncayo, M; Takai, T; Higaki, K; Mena-Barragán, T; Hirano, Y; Yura, K; Li, L; Yu, Y; Ninomiya, H; García-Moreno, M I; Ishii, S; Sakakibara, Y; Ohno, K; Nanba, E; Ortiz Mellet, C; García Fernández, J M; Suzuki, Y

    2012-07-01

    Competitive inhibitors of either α-galactosidase (α-Gal) or β-galactosidase (β-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and β-Gal associated with Fabry disease and GM(1) gangliosidosis. PMID:22618082

  6. Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

    PubMed Central

    Pausch, Patrick; Singh, Ujjwala; Ahmed, Yasar Luqman; Pillet, Benjamin; Murat, Guillaume; Altegoer, Florian; Stier, Gunter; Thoms, Matthias; Hurt, Ed; Sinning, Irmgard; Bange, Gert; Kressler, Dieter

    2015-01-01

    Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation. PMID:26112308

  7. Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

    NASA Astrophysics Data System (ADS)

    Pausch, Patrick; Singh, Ujjwala; Ahmed, Yasar Luqman; Pillet, Benjamin; Murat, Guillaume; Altegoer, Florian; Stier, Gunter; Thoms, Matthias; Hurt, Ed; Sinning, Irmgard; Bange, Gert; Kressler, Dieter

    2015-06-01

    Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

  8. Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture

    PubMed Central

    Kurt, Ramazan; Chandra, Partha K.; Aboulnasr, Fatma; Panigrahi, Rajesh; Ferraris, Pauline; Aydin, Yucel; Reiss, Krzysztof; Wu, Tong; Balart, Luis A.; Dash, Srikanta

    2015-01-01

    -treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA). Conclusion Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1. PMID:25961570

  9. Regional deficiencies in chaperone-mediated autophagy underlie α-synuclein aggregation and neurodegeneration

    PubMed Central

    Malkus, Kristen A.; Ischiropoulos, Harry

    2012-01-01

    In neurodegenerative diseases, it remains unclear why certain brain regions are selectively vulnerable to protein aggregation. In transgenic mice expressing human A53T α-synuclein, the brainstem and spinal cord develop the most prominent α-synuclein inclusions which correlate with age-dependent motor dysfunction. Herein we present the novel finding that this selective aggregation is in part dependent on the inability of chaperone-mediated autophagy (CMA) to effectively degrade α-synuclein in these brain regions. Lysosomal assays revealed that CMA activity was significantly decreased in aggregation-prone regions compared to the remainder of the brain. Previously, CMA activity has been shown to be proportional to levels of the CMA receptor Lamp-2a. Using antibodies, brain tissue from Lamp-2a null mice, enzymatic deglycosylation, and mass spectrometry, we identified Lamp2a as a novel 72 kDa glycoprotein in the mouse brain. Examination of Lamp-2a levels revealed differences in expression across brain regions. The brainstem and the spinal cord had a more than three-fold greater levels of Lamp-2a as compared to regions less vulnerable to aggregation and exhibited a selective upregulation of Lamp-2a during development of α-synuclein inclusions. Despite this dynamic response of Lamp-2a, the levels of substrates bound to the brain lysosomes as well as the rates of substrate uptake and degradation were not proportional to the levels of Lamp-2a. These regional differences in CMA activity and Lamp-2a expression were found in both non-transgenic mice as well as A53T α-syn mice. Therefore, these are inherent variations and not a transgene-specific effect. However, differences in CMA activity may render select brain regions vulnerable to homeostatic dysfunction in the presence of stressors such as overexpression of human A53T α-syn. Collectively, the data provide a potential mechanism to explain the dichotomy of vulnerability or resistance that underlies brain regions

  10. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    SciTech Connect

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V.

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between

  11. The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex*

    PubMed Central

    Trcka, Filip; Durech, Michal; Man, Petr; Hernychova, Lenka; Muller, Petr; Vojtesek, Borivoj

    2014-01-01

    Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding. PMID:24567332

  12. Improvement of chaperone activity of 2-Cys peroxiredoxin using electron beam

    NASA Astrophysics Data System (ADS)

    Hong, Sung Hyun; An, Byung Chull; Lee, Seung Sik; Lee, Jae Taek; Cho, Jae-Hyun; Jung, Hyun Suk; Chung, Byung Yeoup

    2012-08-01

    The peroxiredoxin protein expressed in Pseudomonas aeruginosa PAO1 (PaPrx) is a typical 2-cysteine peroxiredoxin that has dual functions as both a thioredoxin-dependent peroxidase and molecular chaperone. As the function of PaPrx is regulated by its structural status, in the present study, we examined the effects of electron beam radiation on the structural modifications of PaPrx, as well as changes to PaPrx peroxidase and chaperone functions. It was found that the chaperone activity of PaPrx was increased approximately 3- to 4-fold at 2 kGy when compared to non-irradiated PaPrx, while its peroxidase activity decreased. This corresponded to a shift from the low molecular weight PaPrx species that acts as a peroxidase to the high molecular weight complex that functions as a chaperone, as detected using polyacrylamide gel electrophoresis. We also investigated the influence of the electron beam on physical protein properties such as hydrophobicity and secondary structure. The exposure of the PaPrx hydrophobic domains in response to irradiation reached a peak at 2 kGy and then decreased in a dose-dependent manner at higher doses. In addition, the exposure of β-sheet and random coil elements on the surface of PaPrx was significantly increased following irradiation with an electron beam, whereas exposure of α-helix and turn elements was decreased. These results suggest that irradiated PaPrx may be a potential candidate for use in bio-engineering systems and various industrial applications, due to its enhanced chaperone activity.

  13. Mimicking phosphorylation of αB-crystallin affects its chaperone activity

    PubMed Central

    Ecroyd, Heath; Meehan, Sarah; Horwitz, Joseph; Aquilina, J. Andrew; Benesch, Justin L. P.; Robinson, Carol V.; Macphee, Cait E.; Carver, John A.

    2006-01-01

    αB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to αB-crystallin. In the present study, we produced recombi-nant proteins designed to mimic phosphorylation of αB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural invest-igation of the effect of phosphorylation on the chaperone activity of αB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of αB-crystallin results in more efficient chaperone activity against both heat-induced and reduc-tion-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of αB-crystallin against one amyloid-forming target protein (κ-casein), but decreased it against another (ccβ-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated αB-crystallins. The present study provides evidence for the regulation of the chaperone activity of αB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases. PMID:16928191

  14. The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies.

    PubMed

    Luo, B; Lee, A S

    2013-02-14

    Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires glucose regulated protein (GRP) 78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo. Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Because of intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The protein kinase RNA-like ER kinase signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the inositol-requiring kinase 1α branch promotes tumorigenesis, cancer cell survival and regulates tumor invasion. In summary, perturbance of ER homeostasis has critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor

  15. Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

    PubMed Central

    Mahboubi, Hicham

    2015-01-01

    Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities. Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i) the physiological state of the cell and (ii) the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm. Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1. PMID:26713263

  16. The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x

    PubMed Central

    Daniyan, Michael O.; Boshoff, Aileen; Prinsloo, Earl; Pesce, Eva-Rachele; Blatch, Gregory L.

    2016-01-01

    Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria. PMID:26845441

  17. Hydroimidazolone Modification of Human αA-Crystallin: Effect on the Chaperone Function and Protein Refolding Ability

    PubMed Central

    Gangadhariah, Mahesha H.; Wang, Benlian; Linetsky, Mikhail; Henning, Christian; Spanneberg, Robert; Glomb, Marcus A.; Nagaraj, Ram H.

    2010-01-01

    Alpha A-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), αA-crystallin becomes a better chaperone. Alpha A-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of αA-crystallin by MGO (with 20-500 μM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of βL- and γ-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured βL- and γ-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of αA-crystallin to assist in the refolding of denatured MDH. However, MGO-modification of chaperone-null TNS-bound αA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of αA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the αA-crystallin responsible for the chaperone function and refolding are the same in the native αA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance αA-crystallin's ability to refold denatured proteins. PMID:20085807

  18. Knock-down of Hdj2/DNAJA1 co-chaperone results in an unexpected burst of tumorigenicity of C6 glioblastoma cells

    PubMed Central

    Dobrodumov, Anatoliy V.; Komarova, Elena Y.; Voronkina, Irina V.; Lazarev, Vladimir F.; Margulis, Boris A.; Guzhova, Irina V.

    2016-01-01

    The chaperone system based on Hsp70 and proteins of the DnaJ family is known to protect tumor cells from a variety of cytotoxic factors, including anti-tumor therapy. To analyze whether this also functions in a highly malignant brain tumor, we knocked down the expression of Hsp70 (HSPA1A) and its two most abundant co-chaperones, Hdj1 (DNAJB1) and Hdj2 (DNAJA1) in a C6 rat glioblastoma cell line. As expected, tumor depletion of Hsp70 caused a substantial reduction in its growth rate and increased the survival of tumor-bearing animals, whereas the reduction of Hdj1 expression had no effect. Unexpectedly, a reduction in the expression of Hdj2 led to the enhanced aggressiveness of the C6 tumor, demonstrated by its rapid growth, metastasis formation and a 1.5-fold reduction in the lifespan of tumor-bearing animals. The in vitro reduction of Hdj2 expression reduced spheroid density and simultaneously enhanced the migration and invasion of C6 cells. At the molecular level, a knock-down of Hdj2 led to the relocation of N-cadherin and the enhanced activity of metalloproteinases 1, 2, 8 and 9, which are markers of highly malignant cancer cells. The changes in the actin cytoskeleton in Hdj2-depleted cells indicate that the protein is also important for prevention of the amoeboid-like transition of tumor cells. The results of this study uncover a completely new role for the Hdj2 co-chaperone in tumorigenicity and suggest that the protein is a potential drug target. PMID:26959111

  19. Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites.

    PubMed

    Ettrich, Rüdiger; Brandt, Wolfgang; Kopecký, Vladimír; Baumruk, Vladimír; Hofbauerová, Katerina; Pavlícek, Zdenek

    2002-10-01

    With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain. PMID:12452443

  20. Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

    PubMed

    Megidish, T; Takio, K; Titani, K; Iwabuchi, K; Hamaguchi, A; Igarashi, Y; Hakomori, S

    1999-03-16

    Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly

  1. MiR-17-5p Impairs Trafficking of H-ERG K+ Channel Protein by Targeting Multiple ER Stress-Related Chaperones during Chronic Oxidative Stress

    PubMed Central

    Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe

    2013-01-01

    Background To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. Methods We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K+ current. Results H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. Conclusions Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress. PMID:24386440

  2. A novel function of the monomeric CCTε subunit connects the serum response factor pathway to chaperone-mediated actin folding

    PubMed Central

    Elliott, Kerryn L.; Svanström, Andreas; Spiess, Matthias; Karlsson, Roger; Grantham, Julie

    2015-01-01

    Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Although some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCTε, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/serum response factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes after actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCTε subunit by siRNA enhances SRF signaling in cultured mammalian cells by an actin assembly-independent mechanism. Overexpression of CCTε in its monomeric form revealed that CCTε binds via its substrate-binding domain to the C-terminal region of MRTF-A and that CCTε is able to alter the nuclear accumulation of MRTF-A after stimulation by serum addition. Given that the levels of monomeric CCTε conversely reflect the levels of CCT oligomer, our results suggest that CCTε provides a connection between the actin-folding capacity of the cell and actin expression. PMID:26063733

  3. Bag6/Bat3/Scythe: a novel chaperone activity with diverse regulatory functions in protein biogenesis and degradation.

    PubMed

    Lee, Jin-Gu; Ye, Yihong

    2013-04-01

    Upon emerging from the ribosome exiting tunnel, polypeptide folding occurs immediately with the assistance of both ribosome-associated and free chaperones. While many chaperones known to date are dedicated folding catalysts, recent studies have revealed a novel chaperoning system that functions at the interface of protein biogenesis and quality control by using a special "holdase" activity in order to sort and channel client proteins to distinct destinations. The key component, Bag6/Bat3/Scythe, can effectively shield long hydrophobic segments exposed on the surface of a polypeptide, preventing aggregation or inappropriate interactions before a triaging decision is made. The biological consequences of Bag6-mediated chaperoning are divergent for different substrates, ranging from membrane integration to proteasome targeting and destruction. Accordingly, Bag6 can act in various cellular contexts in order to execute many essential cellular functions, while dysfunctions in the Bag6 system can cause severe cellular abnormalities that may be associated with some pathological conditions. PMID:23417671

  4. ATPase Activity and ATP-dependent Conformational Change in the Co-chaperone HSP70/HSP90-organizing Protein (HOP)*

    PubMed Central

    Yamamoto, Soh; Subedi, Ganesh Prasad; Hanashima, Shinya; Satoh, Tadashi; Otaka, Michiro; Wakui, Hideki; Sawada, Ken-ichi; Yokota, Shin-ichi; Yamaguchi, Yoshiki; Kubota, Hiroshi; Itoh, Hideaki

    2014-01-01

    Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change. PMID:24535459

  5. Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection

    PubMed Central

    Verchot, Jeanmarie

    2012-01-01

    Cellular chaperones and folding enzymes play central roles in the formation of positive-strand and negative-strand RNA virus infection. This article examines the key cellular chaperones and discusses evidence that these factors are diverted from their cellular functions to play alternative roles in virus infection. For most chaperones discussed, their primary role in the cell is to ensure protein quality control. They are system components that drive substrate protein folding, complex assembly or disaggregation. Their activities often depend upon co-chaperones and ATP hydrolysis. During plant virus infection, Hsp70 and Hsp90 proteins play central roles in the formation of membrane-bound replication complexes for certain members of the tombusvirus, tobamovirus, potyvirus, dianthovirus, potexvirus, and carmovirus genus. There are several co-chaperones, including Yjd1, RME-8, and Hsp40 that associate with the bromovirus replication complex, pomovirus TGB2, and tospovirus Nsm movement proteins. There are also examples of plant viruses that rely on chaperone systems in the endoplasmic reticulum (ER) to support cell-to-cell movement. TMV relies on calreticulin to promote virus intercellular transport. Calreticulin also resides in the plasmodesmata and plays a role in calcium sequestration as well as glycoprotein folding. The pomovirus TGB2 interacts with RME-8 in the endosome. The potexvirus TGB3 protein stimulates expression of ER resident chaperones via the bZIP60 transcription factor. Up-regulating factors involved in protein folding may be essential to handling the load of viral proteins translated along the ER. In addition, TGB3 stimulates SKP1 which is a co-factor in proteasomal degradation of cellular proteins. Such chaperones and co-factors are potential targets for antiviral defense. PMID:23230447

  6. Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH<3) in E. coli and Shigella spp. Here we investigated the roles of these two acid chaperones in survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, th...

  7. Metal Chaperones: A Holistic Approach to the Treatment of Alzheimer’s Disease

    PubMed Central

    Adlard, Paul Anthony; Bush, Ashley Ian

    2012-01-01

    As evidence for the role of metal ion dysregulation in the pathogenesis of multiple CNS disorders grows, it has become important to more precisely identify and differentiate the biological effects of various pharmacological modulators of metal ion homeostasis. This is particularly evident in disorders such as Alzheimer’s disease (AD), where the use of metal chaperones (that transport metals), as opposed to chelators (which exclude metals from biological interactions), may prove to be the first truly disease modifying approach for this condition. The purpose of this mini-review is to highlight the emerging notion that metal chaperones, such as PBT2 (Prana Biotechnology), modulate a variety of critical pathways affecting key aspects of the AD cascade to provide a more “holistic” approach to the treatment of this disease. PMID:22403554

  8. Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe

    PubMed Central

    Han, Jiyoung; Kim, Kanghwa

    2015-01-01

    To screen molecular chaperones similar to small heat shock proteins (sHsps), but without α-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an α-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70℃ for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no α-crystalline domain in their sequences. PMID:26539043

  9. Structure of the hypothetical Mycoplasma protein, MPN555, suggestsa chaperone function

    SciTech Connect

    Schulze-Gahmen, Ursula; Aono, Shelly; Chen, Shengfeng; Yokota,Hisao; Kim, Rosalind; Kim, Sung-Hou

    2005-06-15

    The crystal structure of the hypothetical protein MPN555from Mycoplasma pneumoniae (gi pbar 1673958) has been determined to a resolution of 2.8 Angstrom using anomalous diffraction data at the Sepeak wavelength. Structure determination revealed a mostly alpha-helical protein with a three-lobed shape. The three lobes or fingers delineate a central binding groove and additional grooves between lobes 1 and 3, and between lobes 2 and 3. For one of the molecules in the asymmetric unit,the central binding pocket was filled with a peptide from the uncleaved N-terminal affinity tag. The MPN555 structure has structural homology to two bacterial chaperone proteins, SurA and trigger factor from Escherichia coli. The structural data and the homology to other chaperone for MPN555.

  10. Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.

    PubMed

    Zierer, Bettina K; Weiwad, Matthias; Rübbelke, Martin; Freiburger, Lee; Fischer, Gunter; Lorenz, Oliver R; Sattler, Michael; Richter, Klaus; Buchner, Johannes

    2014-11-01

    The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors. PMID:25244159

  11. Unfolding the relationship between secreted molecular chaperones and macrophage activation states

    PubMed Central

    Henderson, Samantha

    2008-01-01

    Over the last 20 years, it has emerged that many molecular chaperones and protein-folding catalysts are secreted from cells and function, somewhat in the manner of cytokines, as pleiotropic signals for a variety of cells, with much attention being focused on the macrophage. During the last decade, it has become clear that macrophages respond to bacterial, protozoal, parasitic and host signals to generate phenotypically distinct states of activation. These activation states have been termed ‘classical’ and ‘alternative’ and represent not a simple bifurcation in response to external signals but a range of cellular phenotypes. From an examination of the literature, the hypothesis is propounded that mammalian molecular chaperones are able to induce a wide variety of alternative macrophage activation states, and this may be a system for relating cellular or tissue stress to appropriate macrophage responses to restore homeostatic equilibrium. PMID:18958583

  12. Cobalamin-dependent dehydratases and a deaminase: radical catalysis and reactivating chaperones.

    PubMed

    Toraya, Tetsuo

    2014-02-15

    Adenosylcobalamin, a coenzyme form of vitamin B12, is an organometallic compound that participates in about ten enzymatic reactions. These enzymes catalyze chemically challenging reactions by using a highly reactive primary carbon radical that is derived from homolysis of the coenzyme Co-C bond. Among them, diol dehydratases and ethanolamine ammonia-lyase have been most extensively studied to establish the general mechanism of adenosylcobalamin-assisted enzymatic catalysis and radical-catalyzed reactions. Another important point is that adenosylcobalamin-dependent radical enzymes are prone to mechanism-based irreversible inactivation during catalysis and have their own chaperones for the maintenance of catalytic activities. This review will highlight biochemical, structural, and computational studies with special emphases on radical catalysis and reactivating chaperones of these enzymes. PMID:24269950

  13. Structure of the human histone chaperone FACT Spt16 N-terminal domain.

    PubMed

    Marcianò, G; Huang, D T

    2016-02-01

    The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding. PMID:26841762

  14. The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations

    PubMed Central

    Jeronimo, Célia; Watanabe, Shinya; Kaplan, Craig D.; Peterson, Craig L.; Robert, François

    2015-01-01

    SUMMARY H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nucleosome reassembly coupled to pervasive incorporation of H2A.Z by mislocalized SWR-C alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z confinement is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. PMID:25959393

  15. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  16. Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

    PubMed Central

    Ribeiro, Raquel; Gilberto, Samuel; Gomes, Ricardo A.; Ferreira, António; Mateus, Élia; Barroso, Eduardo; Coelho, Ana V.; Freire, Ana Ponces; Cordeiro, Carlos

    2015-01-01

    Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis. PMID:26147092

  17. Bioactive Metabolites from Chaetomium aureum: Structure Elucidation and Inhibition of the Hsp90 Machine Chaperoning Activity

    PubMed Central

    Kabbaj, Fatima Zahra; Lu, Su; Faouzi, My El Abbés; Meddah, Bouchra; Proksch, Peter; Cherrah, Yahya; Altenbach, Hans-Josef; Aly, Amal H.; Chadli, Ahmed; Debbab, Abdessamad

    2014-01-01

    Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48h. PMID:25482429

  18. DJ-1 Is a Redox-Dependent Molecular Chaperone That Inhibits α-Synuclein Aggregate Formation

    PubMed Central

    2004-01-01

    Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to α-synuclein, a protein implicated in PD pathogenesis. PMID:15502874

  19. The molecular chaperone Brichos breaks the catalytic cycle that generates toxic Aβ oligomers

    PubMed Central

    Kurudenkandy, Firoz Roshan; Biverstal, Henrik; Dolfe, Lisa; Dunning, Christopher; Yang, Xiaoting; Frohm, Birgitta; Vendruscolo, Michele; Johansson, Jan; Dobson, Christopher M.; Fisahn, André; Knowles, Tuomas P. J.; Linse, Sara

    2015-01-01

    Alzheimer’s disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces strongly catalyse the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a Brichos domain, can specifically inhibit this catalytic cycle and limit Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living brain tissue by means of cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation. PMID:25686087

  20. Structure of Glycerol Dehydratase Reactivase: A New Type of Molecular Chaperone

    SciTech Connect

    Liao, Der-Ing; Reiss, Lisa; Turner, Jr., Ivan; Dotson, Garry

    2010-03-08

    The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B{sub 12} from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 {angstrom} resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa {alpha} subunits and two globular 14 kDa {beta} subunits. The {alpha} subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the {beta} subunit resembles that of the {beta} subunit of glycerol dehydratase, except that it lacks some coenzyme B12 binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.

  1. Structure of CfaA Suggests a New Family of Chaperones Essential for Assembly of Class 5 Fimbriae

    PubMed Central

    Bao, Rui; Fordyce, April; Chen, Yu-Xing; McVeigh, Annette; Savarino, Stephen J.; Xia, Di

    2014-01-01

    Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly. PMID:25122114

  2. Structure of CfaA suggests a new family of chaperones essential for assembly of class 5 fimbriae.

    PubMed

    Bao, Rui; Fordyce, April; Chen, Yu-Xing; McVeigh, Annette; Savarino, Stephen J; Xia, Di

    2014-08-01

    Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly. PMID:25122114

  3. Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

    PubMed

    Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

    2012-07-01

    Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. PMID:22317751

  4. Role of cysteine residues in the enhancement of chaperone function in methylglyoxal-modified human αA-crystallin

    PubMed Central

    Kanade, Santosh R.; Pasupuleti, NagaRekha

    2013-01-01

    We have previously demonstrated that the reaction of a physiological dicarbonyl, methylglyoxal (MGO) enhances the chaperone function of human αA-crystallin. MGO can react with cysteine, arginine, and lysine residues in proteins. Although the role of arginine and lysine residues in the enhancement of chaperone function has been investigated, the role of cysteine residues is yet to be determined. In this study, we have investigated the effect of MGO modification on the structure and chaperone function of αA-crystallin mutant proteins in which C131 and C142 were replaced either individually or simultaneously with isoleucine. MGO-modification resulted in improved chaperone function in all three αA-crystallin mutants, including the cysteine-free double mutant. The enhanced chaperone function was due to increased surface hydrophobicity and increased binding of client proteins. These results suggest that the two cysteine residues, even though they could be modified, do not take part in the MGO-induced improvement in the chaperone function of human αA-crystallin. PMID:19020808

  5. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    PubMed

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  6. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    PubMed Central

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in-vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  7. Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

    PubMed Central

    Liu, Hongde; Zhu, Min; Mu, Yawen; Liu, Lingjie; Li, Guanghui; Wan, Yakun

    2014-01-01

    Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)2 tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin. PMID:25338502

  8. Oligomeric assembly is required for chaperone activity of the filamentous γ-prefoldin.

    PubMed

    Glover, Dominic J; Clark, Douglas S

    2015-08-01

    Prefoldins (PFDs) are molecular chaperones with a distinctive jellyfish-shape that have a general role in de novo protein folding in Archaea and in the biogenesis of cytoskeleton proteins in eukaryotes. In general, PFDs are hetero-hexameric protein assemblies consisting of two α and four β subunits. However, a PFD variant called gamma-prefoldin (γPFD), isolated from the hyperthermophilic archaeon Methanocaldococcus jannaschii, exhibits a unique filamentous structure that is composed of hundreds of monomeric subunits. In this study, we investigated the relationship between the morphology of the γPFD filament and its ability to prevent protein aggregation. A chaperone assay demonstrated that γPFD must be in a filamentous assembly for functional activity and the distal regions of the coiled-coils are required for binding of non-native proteins. Molecular dynamic simulations were used to model the interactions between in silico thermally denatured protein substrates and the coiled-coils of a γPFD filament. During molecular dynamic simulations at 300 and 353 K, each coiled-coil was highly flexible, enabling it to widen the central cavity of the filament to potentially capture various non-native proteins. Docking molecular dynamic simulations of γPFD filaments with unfolded citrate synthase or insulin showed a size-dependence between the substrate and the number of interacting coiled-coils. To confirm this observation, we generated filaments containing specific numbers of subunits, and showed that between six and eight γPFD subunits are required for chaperone activity to prevent citrate synthase from thermal aggregation. These results provide insights into structure-function relationships of oligomeric chaperones and illuminate the potential role of γPFD in its native environment. PMID:26096656

  9. Pyrimidinone-Peptoid Hybrid Molecules with Distinct Effects on Molecular Chaperone Function and Cell Proliferation

    PubMed Central

    Wright, Christine M.; Chovatiya, Raj J.; Jameson, Nora E.; Turner, David M.; Zhu, Guangyu; Werner, Stefan; Huryn, Donna M.; Pipas, James M.; Day, Billy W.; Wipf, Peter; Brodsky, Jeffrey L.

    2008-01-01

    The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak, but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70. PMID:18164205

  10. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  11. Discovery of Benzisoxazoles as Potent Inhibitors of Chaperone Heat Shock Protein 90

    SciTech Connect

    Gopalsamy, Ariamala; Shi, Mengxiao; Golas, Jennifer; Vogan, Erik; Jacob, Jaison; Johnson, Mark; Lee, Frederick; Nilakantan, Ramaswamy; Petersen, Roseann; Svenson, Kristin; Chopra, Rajiv; Tam, May S.; Wen, Yingxia; Ellingboe, John; Arndt, Kim; Boschelli, Frank

    2008-08-11

    Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

  12. Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96.

    PubMed

    Wang, Jin; Grishin, Anatoly V; Ford, Henri R

    2016-06-15

    Semapimod, a tetravalent guanylhydrazone, suppresses inflammatory cytokine production and has potential in a variety of inflammatory and autoimmune disorders. The mechanism of action of Semapimod is not well understood. In this study, we demonstrate that in rat IEC-6 intestinal epithelioid cells, Semapimod inhibits activation of p38 MAPK and NF-κB and induction of cyclooxygenase-2 by TLR ligands, but not by IL-1β or stresses. Semapimod inhibits TLR4 signaling (IC50 ≈0.3 μmol) and acts by desensitizing cells to LPS; it fails to block responses to LPS concentrations of ≥5 μg/ml. Inhibition of TLR signaling by Semapimod is almost instantaneous: the drug is effective when applied simultaneously with LPS. Semapimod blocks cell-surface recruitment of the MyD88 adapter, one of the earliest events in TLR signaling. gp96, the endoplasmic reticulum-localized chaperone of the HSP90 family critically involved in the biogenesis of TLRs, was identified as a target of Semapimod using ATP-desthiobiotin pulldown and mass spectroscopy. Semapimod inhibits ATP-binding and ATPase activities of gp96 in vitro (IC50 ≈0.2-0.4 μmol). On prolonged exposure, Semapimod causes accumulation of TLR4 and TLR9 in perinuclear space, consistent with endoplasmic reticulum retention, an anticipated consequence of impaired gp96 chaperone function. Our data indicate that Semapimod desensitizes TLR signaling via its effect on the TLR chaperone gp96. Fast inhibition by Semapimod is consistent with gp96 participating in high-affinity sensing of TLR ligands in addition to its role as a TLR chaperone. PMID:27194788

  13. Amphiphilic 1-deoxynojirimycin derivatives through click strategies for chemical chaperoning in N370S Gaucher cells.

    PubMed

    Diot, Jennifer D; Garcia Moreno, Isabel; Twigg, Gabriele; Ortiz Mellet, Carmen; Haupt, Karsten; Butters, Terry D; Kovensky, José; Gouin, Sébastien G

    2011-10-01

    In Gaucher disease (GD), mutant β-glucocerebrosidases (β-GCase) that are misfolded are recognized by the quality control machinery of the endoplasmic reticulum (ER) and degraded proteolytically. Hydrophobic iminosugars can be used as pharmacological chaperones to provide an improvement in the folding of the enzyme and promote trafficking from the ER. We have developed here an efficient click procedure to tether hydrophobic substituents to N-azidopropyl-1-deoxynojirimycin. A set of 14 original iminosugars was designed and evaluated for inhibition of commercially available glucosidases. Most of the compounds were micromolar inhibitors of those enzymes. In vitro inhibition assays with the N370S β-GCase revealed that the sublibrary containing the derivatives with aromatic aglycons displayed the highest inhibitory potency. Chaperone activity of the whole set of synthetic compounds was also explored in mutant Gaucher cells. The most active compound gave a nearly 2-fold increase in enzyme activity at 20 μM, a significantly higher value than the 1.33-fold recorded for the reference compound N-nonyl-1-deoxynojirimycin (N-nonyl-DNJ). As previously reported with bicyclic sp(2)-iminosugars (Luan, Z.; Higaki, K.; Aguilar-Moncayo, M.; Ninomiya, H.; Ohno, K.; García-Moreno, M. I.; Ortiz Mellet, C.; García Fernández, J. M.; Suzuki, Y. ChemBioChem 2009, 10, 2780), in vitro inhibition of β-GCase measured for the compounds did not correlate with the cellular chaperone activity. The potency of new iminosugar chaperones is therefore not predictable from structure-activity relationships studies based on the in vitro β-GCase inhibition. PMID:21830816

  14. Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Chaperones Sharing Mitochondrial Enzymes as Clients

    PubMed Central

    Erives, Albert J.; Fassler, Jan S.

    2015-01-01

    The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones. PMID:25710177

  15. Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    PubMed Central

    Trisciuoglio, Daniela; Gabellini, Chiara; Desideri, Marianna; Ziparo, Elio; Zupi, Gabriella; Del Bufalo, Donatella

    2010-01-01

    Background Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. Methodology/Principal Findings By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon. Conclusions/Significance We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the

  16. Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy.

    PubMed

    Ulbricht, Anna; Eppler, Felix J; Tapia, Victor E; van der Ven, Peter F M; Hampe, Nico; Hersch, Nils; Vakeel, Padmanabhan; Stadel, Daniela; Haas, Albert; Saftig, Paul; Behrends, Christian; Fürst, Dieter O; Volkmer, Rudolf; Hoffmann, Bernd; Kolanus, Waldemar; Höhfeld, Jörg

    2013-03-01

    Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation. PMID:23434281

  17. The neural chaperone proSAAS blocks α-synuclein fibrillation and neurotoxicity.

    PubMed

    Jarvela, Timothy S; Lam, Hoa A; Helwig, Michael; Lorenzen, Nikolai; Otzen, Daniel E; McLean, Pamela J; Maidment, Nigel T; Lindberg, Iris

    2016-08-01

    Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson's disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson's disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158-180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral dopaminergic neurons, and recombinant proSAAS blocks α-synuclein-induced cytotoxicity in SH-SY5Y cells. Four independent proteomics studies have previously identified proSAAS as a potential cerebrospinal fluid biomarker in various neurodegenerative diseases. Coupled with prior work showing that proSAAS blocks β-amyloid aggregation into fibrils, this study supports the idea that neuronal proSAAS plays an important role in proteostatic processes. ProSAAS thus represents a possible therapeutic target in neurodegenerative disease. PMID:27457957

  18. Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

    PubMed Central

    Boczek, Edgar E.; Reefschläger, Lasse G.; Dehling, Marco; Struller, Tobias J.; Häusler, Elisabeth; Seidl, Andreas; Kaila, Ville R. I.; Buchner, Johannes

    2015-01-01

    Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated. PMID:26056257

  19. Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

    PubMed

    Hossain, Mohammad Arif; Higaki, Katsumi; Saito, Seiji; Ohno, Kazuki; Sakuraba, Hitoshi; Nanba, Eiji; Suzuki, Yoshiyuki; Ozono, Keiichi; Sakai, Norio

    2015-09-01

    Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant GM1 β-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations. PMID:26108143

  20. Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity

    PubMed Central

    Park, Chang-Jin; Seo, Young-Su

    2015-01-01

    As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells. PMID:26676169

  1. Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq.

    PubMed

    Shiratsuchi, Akiko; Nitta, Mao; Kuroda, Ayumi; Komiyama, Chiharu; Gawasawa, Mitsuko; Shimamoto, Naoto; Tuan, Tran Quoc; Morita, Teppei; Aiba, Hiroji; Nakanishi, Yoshinobu

    2016-08-15

    An RNA chaperone of Escherichia coli, called host factor required for phage Qβ RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity. PMID:27357148

  2. Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

    PubMed

    Kimmins, S; MacRae, T H

    2000-04-01

    The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding. PMID:11147968

  3. Structures of GRP94-Nucleotide Complexes Reveal Mechanistic Differences Between the Hsp90 Chaperones

    SciTech Connect

    Dollins, D.E.; Warren, J.J.; Immormino, R.M.; Gewirth, D.T.

    2009-06-02

    GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 {angstrom} crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a 'twisted V' conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.

  4. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A.

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  5. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  6. TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon.

    PubMed

    Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig

    2015-10-20

    TRP channels and G protein-coupled receptors (GPCRs) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which, in turn, associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832

  7. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  8. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    NASA Astrophysics Data System (ADS)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  9. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  10. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

    PubMed Central

    Wagner, I; Arlt, H; van Dyck, L; Langer, T; Neupert, W

    1994-01-01

    ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria. Images PMID:7957078

  11. Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism.

    PubMed

    Schulze, Andrea; Beliu, Gerti; Helmerich, Dominic A; Schubert, Jonathan; Pearl, Laurence H; Prodromou, Chrisostomos; Neuweiler, Hannes

    2016-08-01

    The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle. PMID:27322067

  12. The endoplasmic reticulum protein folding factory and its chaperones: new targets for drug discovery?

    PubMed Central

    McLaughlin, Martin; Vandenbroeck, Koen

    2011-01-01

    Cytosolic heat shock proteins have received significant attention as emerging therapeutic targets. Much of this excitement has been triggered by the discovery that HSP90 plays a central role in the maintenance and stability of multifarious oncogenic membrane receptors and their resultant tyrosine kinase activity. Numerous studies have dealt with the effects of small molecules on chaperone- and stress-related pathways of the endoplasmic reticulum (ER). However, unlike cytosolic chaperones, relatively little emphasis has been placed upon translational avenues towards targeting of the ER for inhibition of folding/secretion of disease-promoting proteins. Here, we summarise existing small molecule inhibitors and potential future targets of ER chaperone-mediated inhibition. Client proteins of translational relevance in disease treatment are outlined, alongside putative future disease treatment modalities based on ER-centric targeted therapies. Particular attention is paid to cancer and autoimmune disorders via the effects of the GRP94 inhibitor geldanamycin and its population of client proteins, overloading of the unfolded protein response, and inhibition of members of the IL-12 family of cytokines by celecoxib and non-coxib analogues. PMID:20942857

  13. Crystallization of the FaeE chaperone of Escherichia coli F4 fimbriae

    SciTech Connect

    Van Molle, Inge Buts, Lieven; Coppens, Fanny; Qiang, Liu; Wyns, Lode; Loris, Remy; Bouckaert, Julie; De Greve, Henri

    2005-04-01

    The periplasmic chaperone FaeE of E. coli F4 fimbriae crystallizes in three crystal forms. F4 (formerly K88) fimbriae from enterotoxigenic Escherichia coli are assembled via the FaeE/FaeD chaperone/usher pathway. The chaperone FaeE crystallizes in three crystal forms, all belonging to space group C2. Crystals of form 1 diffract to 2.3 Å and have unit-cell parameters a = 195.7, b = 78.5, c = 184.6 Å, β = 102.2°. X-ray data for crystal form 2 were collected to 2.7 Å using an SeMet variant of FaeE. The crystals have unit-cell parameters a = 136.4, b = 75.7, c = 69.4 Å, β = 92.8°. Crystals of form 3 were formed in a solution containing the FaeE–FaeG complex and diffract to 2.8 Å. Unit-cell parameters are a = 109.7, b = 78.6, c = 87.8 Å, β = 96.4°.

  14. Celastrol increases glucocerebrosidase activity in Gaucher disease by modulating molecular chaperones

    PubMed Central

    Yang, Chunzhang; Swallows, Cody L.; Zhang, Chao; Lu, Jie; Xiao, Hongbin; Brady, Roscoe O.; Zhuang, Zhengping

    2014-01-01

    Gaucher disease is caused by mutations in the glucosidase, beta, acid gene that encodes glucocerebrosidase (GCase). Glucosidase, beta, acid mutations often cause protein misfolding and quantitative loss of GCase. In the present study, we found that celastrol, an herb derivative with known anticancer, anti-inflammatory, and antioxidant activity, significantly increased the quantity and catalytic activity of GCase. Celastrol interfered with the establishment of the heat-shock protein 90/Hsp90 cochaperone Cdc37/Hsp90-Hsp70-organizing protein chaperone complex with mutant GCase and reduced heat-shock protein 90-associated protein degradation. In addition, celastrol modulated the expression of molecular chaperones. Bcl2-associated athanogene 3 and heat shock 70kDa proteins 1A and 1B were significantly increased by celastrol. Furthermore, BAG family molecular chaperone regulator 3 assisted protein folding and maturation of mutant GCase. These findings provide insight into a therapeutic strategy for Gaucher disease and other human disorders that are associated with protein misfolding. PMID:24351928

  15. Pharmacological Chaperone Design for Reducing Risk Factor of Parkinson's Disease from Traditional Chinese Medicine

    PubMed Central

    Huang, Hung-Jin; Lee, Cheng-Chun; Chen, Calvin Yu-Chian

    2014-01-01

    Dysfunction of β-glucocerebrosidase (GCase) has no hydrolytic activity in patients of Gaucher's disease and increasing the risk factor for Parkinson's disease occurrence. Pharmacological chaperone design has been used to treat with misfolded protein in related disease, which utilized a small compound to cause protein folding correctly. This study employed the world largest traditional Chinese medicine (TCM) database for searching for potential lead compound as pharmacological chaperone, and we also performed molecular dynamics (MD) simulations to observe the stability of binding conformation between ligands and active site of GCase structure. The docking results from database screening show that N-methylmescaline and shihunine have high binding ability to GCase than tetrahydroxyazepanes. From MD simulation analysis, tetrahydroxyazepanes displayed high opportunity of ligand migration instead of our TCM candidates, and H-bonds number was decreased in the end of MD snapshot. Our result indicated that binding conformation of N-methylmescaline and shihunine remains stable during MD simulation, demonstrating that the two candidates are suitable for GCase binding and might be potential as pharmacological chaperone for GCase folding correctly. PMID:24527054

  16. TRP and rhodopsin transport depends on dual XPORT ER chaperones encoded by an operon

    PubMed Central

    Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig

    2015-01-01

    Summary TRP channels and G protein-coupled receptors (GPCR) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic, and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which in turn associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832

  17. N. meningitidis 1681 is a member of the FinO family of RNA chaperones.

    SciTech Connect

    Chaulk, S.; Lu, J.; Tan, K.; Arthur, D.; Edwards, R.; Frost, L.; Joachimiak, A.; Glover, J.

    2010-11-01

    The conjugative transfer of F-like plasmids between bacteria is regulated by the plasmid-encoded RNA chaperone, FinO, which facilitates sense - antisense RNA interactions to regulate plasmid gene expression. FinO was thought to adopt a unique structure, however many putative homologs have been identified in microbial genomes and are considered members of the FinO-conjugation-repressor superfamily. We were interested in determining whether other members were also able to bind RNA and promote duplex formation, suggesting that this motif does indeed identify a putative RNA chaperone. We determined the crystal structure of the N. meningitidis MC58 protein NMB1681. It revealed striking similarity to FinO, with a conserved fold and a large, positively charged surface that could function in RNA interactions. Using assays developed to study FinO-FinP sRNA interactions, NMB1681, like FinO, bound tightly to FinP RNA stem-loops with short 5-foot and 3-foot single-stranded tails but not to ssRNA. It also was able to catalyze strand exchange between an RNA duplex and a complementary single-strand, and facilitated duplexing between complementary RNA hairpins. Finally, NMB1681 was able to rescue a finO deficiency and repress F plasmid conjugation. This study strongly suggests that NMB1681 is a FinO-like RNA chaperone that likely regulates gene expression through RNA-based mechanisms in N. meningitidis.

  18. The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

    PubMed Central

    Jahn, Markus; Rehn, Alexandra; Pelz, Benjamin; Hellenkamp, Björn; Richter, Klaus; Rief, Matthias; Buchner, Johannes; Hugel, Thorsten

    2014-01-01

    The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90’s chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90’s chaperone cycle governing client activation, cell viability, and stress tolerance. PMID:25468961

  19. Maf-dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export

    PubMed Central

    Parker, Jennifer L; Lowry, Rebecca C; Couto, Narciso A S; Wright, Phillip C; Stafford, Graham P; Shaw, Jonathan G

    2014-01-01

    Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form. PMID:24527847

  20. A review of acquired thermotolerance, heat shock proteins, and molecular chaperones in archaea

    SciTech Connect

    Trent, J.D.

    1996-05-01

    Acquired thermotolerance, the associated synthesis of heat-shock proteins (HSPs) under stress conditions, and the role of HSPs as molecular chaperones under normal growth conditions have been studied extensively in eukaryotes and bacteria, whereas research in these areas in archaea is only beginning. All organisms have evolved a variety of strategies for coping with high-temperature stress, and among these strategies is the increased synthesis of HSPs. The facts that both high temperatures and chemical stresses induce the HSPs and that some of the HSPs recognize and bind to unfolded proteins in vitro have led to the theory that the function of HSPs is to prevent protein aggregation in vivo. The facts that some HSPs are abundant under normal growth conditions and that they assist in protein folding in vitro have led to the theory that they assist protein folding in vivo; in this role, they are referred to as molecular chaperones. The limited research on acquired thermotolerance, HSPs, and molecular chaperones in archaea, particularly the hyperthermophilic archaea, suggests that these extremophiles provide a new perspective in these areas of research, both because they are members of a separate phylogenetic domain and because they have evolved to live under extreme conditions.

  1. Activation of RidA chaperone function by N-chlorination

    PubMed Central

    Müller, Alexandra; Langklotz, Sina; Lupilova, Nataliya; Kuhlmann, Katja; Bandow, Julia Elisabeth; Leichert, Lars Ingo Ole

    2014-01-01

    Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress. PMID:25517874

  2. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    PubMed Central

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  3. Mitochondrial import receptors Tom20 and Tom22 have chaperone-like activity.

    PubMed

    Yano, Masato; Terada, Kazutoyo; Mori, Masataka

    2004-03-12

    Mitochondrial preproteins are synthesized in the cytosol with N-terminal signal sequences (presequences) or internal targeting signals. Generally, preproteins with presequences are initially recognized by Tom20 (translocase of the outer membrane) and, subsequently, by Tom22, whereas hydrophobic preproteins with internal targeting signals are first recognized by Tom70. Recent studies suggest that Tom70 associates with molecular chaperones, thereby maintaining their substrate preproteins in an import-competent state. However, such a function has not been reported for other Tom component(s). Here, we investigated a role for Tom20 in preventing substrate preproteins from aggregating. In vitro binding assays showed that Tom20 binds to guanidinium chloride unfolded substrate proteins regardless of the presence or absence of presequences. This suggests that Tom20 functions as a receptor not only for presequences but also for mature portions exposed in unfolded preproteins. Aggregation suppression assays on citrate synthase showed that the cytosolic domain of Tom20 has a chaperone-like activity to prevent this protein from aggregating. This activity was inhibited by a presequence peptide, suggesting that the binding site of Tom20 for presequence is identical or close to the active site for the chaperone-like activity. The cytosolic domain of Tom22 also showed a similar activity for citrate synthase, whereas Tom70 did not. These results suggest that the cytosolic domains of Tom20 and Tom22 function to maintain their substrate preproteins unfolded and prevent them from aggregating on the mitochondrial surface. PMID:14699115

  4. Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes

    PubMed Central

    Angelov, Dimitar; Bondarenko, Vladimir A; Almagro, Sébastien; Menoni, Hervé; Mongélard, Fabien; Hans, Fabienne; Mietton, Flore; Studitsky, Vasily M; Hamiche, Ali; Dimitrov, Stefan; Bouvet, Philippe

    2006-01-01

    Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A–H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A–H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription. PMID:16601700

  5. The p23 co-chaperone protein is a novel substrate of CK2 in Arabidopsis.

    PubMed

    Tosoni, Kendra; Costa, Alex; Sarno, Stefania; D'Alessandro, Stefano; Sparla, Francesca; Pinna, Lorenzo A; Zottini, Michela; Ruzzene, Maria

    2011-10-01

    The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses. PMID:21735091

  6. The co-chaperone p23 controls root development through the modulation of auxin distribution in the Arabidopsis root meristem

    PubMed Central

    D’Alessandro, Stefano; Golin, Serena; Hardtke, Christian S.; Lo Schiavo, Fiorella

    2015-01-01

    Homologues of the p23 co-chaperone of HSP90 are present in all eukaryotes, suggesting conserved functions for this protein throughout evolution. Although p23 has been extensively studied in animal systems, little is known about its function in plants. In the present study, the functional characterization of the two isoforms of p23 in Arabidopsis thaliana is reported, suggesting a key role of p23 in the regulation of root development. Arabidopsis p23 mutants, for either form, show a short root length phenotype with a reduced meristem length. In the root meristem a low auxin level associated with a smaller auxin gradient was observed. A decrease in the expression levels of PIN FORMED PROTEIN (PIN)1, PIN3, and PIN7, contextually to an inefficient polar localization of PIN1, was detected. Collectively these results suggest that both Arabidopsis p23 isoforms are required for root growth, in particular in the maintenance of the root meristem, where the proteins are located. PMID:26163704

  7. Overexpression of CHOP alone and in combination with chaperones is effective in improving antibody production in mammalian cells.

    PubMed

    Nishimiya, Daisuke; Mano, Takashi; Miyadai, Kenji; Yoshida, Hiroko; Takahashi, Tohru

    2013-03-01

    Secretory capacities including folding and assembly are believed to be limiting factors in the establishment of mammalian cell lines producing high levels of recombinant therapeutic proteins. To achieve industrial success, it is also important to improve protein folding, assembly, and secretory processes in combination with increasing transcription and translation. Here, we identified the expression of CHOP/Gadd153 and GRP78, which are unfolded protein response (UPR)-related genes, correlated with recombinant antibody production in stable CHO cells. Subsequently, CHOP overexpression resulted in increasing recombinant antibody production in some mammalian cell lines, and in addition a threefold further enhancement was obtained by combining expression with UPR-related genes or ER chaperones in transient assays. Overexpression of CHOP had no effect on the biochemical characteristics of the product. These results suggest overexpression of CHOP and its combinations may be an effective method to efficiently select a single cell line with a high level of antibody production in the development of cell lines for manufacturing. PMID:22926643

  8. Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease.

    PubMed

    Jung, Olive; Patnaik, Samarjit; Marugan, Juan; Sidransky, Ellen; Westbroek, Wendy

    2016-05-01

    Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones. PMID:27098312

  9. The molecular chaperone system and other anti-stress mechanisms in archaea.

    PubMed

    Macario, A J; Conway De Macario, E

    2001-02-01

    This article presents a brief review of stressors, their cellular and intracellular targets, stress proteins, molecular chaperones, and other anti-stress mechanisms. New data are reported on cochaperones and multicellular structures in archaea. The molecular chaperoning systems of bacteria and eukaryotes have been studied for many years and are relatively well known in terms of their components and mechanisms of action, although many details remain to be elucidated and almost certainly other components will be discovered in the future. By comparison, the molecular chaperoning system of archaea is still unexplored. Since archaea have some molecular genetic and physiologic features similar to those of bacteria and some resembling those of eukaryotes, extrapolation from what is known of organisms from these two phylogenetic domains to archaeal species is unwarranted. For example, the components of the molecular chaperone machine, Hsp70(DnaK), Hsp40(DnaJ), and GrpE, in the archaeal species that have it, are closely related to bacterial counterparts, whereas the archaeal chaperonins are like the eukaryotic equivalents. Furthermore, many archaeal species lack the chaperone machine, in contrast to bacteria and eukaryotes that have it without any known exception. A search for the cochaperones trigger factor, Hop, Hip, BAG-1, and NAC in archaeal genomes demonstrated no conserved equivalents, but two families of archaeal molecules were identified that might be related to NAC and Hop, respectively. Multicellular structures with a single species such as packet and lamina are formed by Methanosarcina species, among which the best studied is M. mazeii. Multispecies multicellular structures are formed by a variety of archaeal organisms, which are either flat (biofilm) or globular (granule) and constitute a functional association or consortium. Details of morphology, formation, and internal organization are described for representative examples of multicellular structures. These

  10. Restoring assembly and activity of cystathionine β-synthase mutants by ligands and chemical chaperones

    PubMed Central

    Kopecká, Jana; Krijt, Jakub; Raková, Kateřina

    2010-01-01

    Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine β-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)—a heme precursor—and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria. Electronic supplementary material The online version of this article (doi:10.1007/s10545-010-9087-5) contains supplementary material, which is available to authorized users. PMID:20490928

  11. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

    PubMed

    Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F; Oliveira, Solange

    2016-01-01

    The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

  12. Cadmium Induces the Expression of Grp78, an Endoplasmic Reticulum Molecular Chaperone, in LLC-PK1 Renal Epithelial Cells

    PubMed Central

    Liu, Fang; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato

    2006-01-01

    To reveal the effects of cadmium exposure on the endoplasmic reticulum (ER) stress response, we examined the expression and function of 78-kDa glucose-regulated protein (Grp78), an ER-resident molecular chaperone, in LLC-PK1 cells. In cells treated with 10 μM cadmium chloride, Grp78 protein levels increased after 6 hr and remained elevated at 24 hr. When cells were incubated with 1–20 μM CdCl2 for 6 hr, Grp78 increased in a dose-dependent manner. In addition, Grp78 mRNA levels were elevated in response to CdCl2 exposure. After exposure to 10 μM CdCl2, the levels of activating transcription factor 4 (ATF4) were increased at 2 hr, with a further enhancement after that; this accumulation followed the transient but marked phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) on serine 51. Although ATF4 mRNA levels increased mildly by CdCl2 exposure, treatment with actinomycin D did not suppress CdCl2-induced accumulation of ATF4 protein, suggesting the involvement of posttranscriptional and, in part, transcriptional mechanisms. Compared with other heavy-metal compounds such as manganese chloride, zinc chloride, mercuric chloride, and lead chloride, CdCl2 could increase the levels of Grp78, ATF4, and the phosphorylated form of eIF2α more markedly without definite cellular damage. The silencing of Grp78 expression using short-interference RNA enhanced CdCl2-induced cellular damage. These results show that cadmium induces the expression of Grp78 probably via phosphorylation of eIF2α and resultant translation of ATF4, and this ER stress response plays a role in protection against cadmium cytotoxicity in this renal epithelial cell. PMID:16759985

  13. Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells.

    PubMed

    Liu, Fang; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato

    2006-06-01

    To reveal the effects of cadmium exposure on the endoplasmic reticulum (ER) stress response, we examined the expression and function of 78-kDa glucose-regulated protein (Grp78) , an ER-resident molecular chaperone, in LLC-PK1 cells. In cells treated with 10 microM cadmium chloride, Grp78 protein levels increased after 6 hr and remained elevated at 24 hr. When cells were incubated with 1-20 microM CdCl2 for 6 hr, Grp78 increased in a dose-dependent manner. In addition, Grp78 mRNA levels were elevated in response to CdCl2 exposure. After exposure to 10 microM CdCl2, the levels of activating transcription factor 4 (ATF4) were increased at 2 hr, with a further enhancement after that ; this accumulation followed the transient but marked phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2(alpha)) on serine 51. Although ATF4 mRNA levels increased mildly by CdCl2 exposure, treatment with actinomycin D did not suppress CdCl2-induced accumulation of ATF4 protein, suggesting the involvement of posttranscriptional and, in part, transcriptional mechanisms. Compared with other heavy-metal compounds such as manganese chloride, zinc chloride, mercuric chloride, and lead chloride, CdCl2 could increase the levels of Grp78, ATF4, and the phosphorylated form of eIF2(alpha) more markedly without definite cellular damage. The silencing of Grp78 expression using short-interference RNA enhanced CdCl2-induced cellular damage. These results show that cadmium induces the expression of Grp78 probably via phosphorylation of eIF2(alpha) and resultant translation of ATF4, and this ER stress response plays a role in protection against cadmium cytotoxicity in this renal epithelial cell. PMID:16759985

  14. Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

    PubMed Central

    Alvarez-Erviti, L; Seow, Y; Schapira, A HV; Rodriguez-Oroz, M C; Obeso, J A; Cooper, J M

    2013-01-01

    The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson's disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis. PMID:23492776

  15. Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells.

    PubMed

    Ishima, Tamaki; Fujita, Yuko; Hashimoto, Kenji

    2014-03-15

    The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect. PMID:24508523

  16. Nucleolar DEAD-Box RNA Helicase TOGR1 Regulates Thermotolerant Growth as a Pre-rRNA Chaperone in Rice

    PubMed Central

    Tang, Ding; Zhang, Yu’e; Cheng, Zhukuan; Xue, Yongbiao

    2016-01-01

    Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs. PMID:26848586

  17. The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog.

    PubMed

    Shafie, Intan N F; McLaughlin, Mark; Burchmore, Richard; Lim, Mary Ann A; Montague, Paul; Johnston, Pamela E J; Penderis, Jacques; Anderson, Thomas J

    2014-05-01

    Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent

  18. Cloning Expression Purification Crystallization and Preliminary X-ray Diffractino Studies of a 12R-LOX-chaperone Complex

    SciTech Connect

    G Deb; K Boeshanes; W Idler; B Ahvazi

    2011-12-31

    Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2{sub 1}, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 {angstrom}, {beta} = 101.07{sup o}. Based on the calculated Matthews coefficient (3.1 {angstrom}3 Da{sup -1}), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 {angstrom} resolution using synchrotron radiation.

  19. Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer-membrane proteins.

    PubMed

    Thoma, Johannes; Burmann, Björn M; Hiller, Sebastian; Müller, Daniel J

    2015-10-01

    Chaperones increase the folding yields of soluble proteins by suppressing misfolding and aggregation, but how they modulate the folding of integral membrane proteins is not well understood. Here we use single-molecule force spectroscopy and NMR spectroscopy to observe the periplasmic holdase chaperones SurA and Skp shaping the folding trajectory of the large β-barrel outer-membrane receptor FhuA from Escherichia coli. Either chaperone prevents FhuA from misfolding by stabilizing a dynamic, unfolded state, thus allowing the substrate to search for structural intermediates. During this search, the SurA-chaperoned FhuA polypeptide inserts β-hairpins into the membrane in a stepwise manner until the β-barrel is folded. The membrane acts as a free-energy sink for β-hairpin insertion and physically separates transient folds from chaperones. This stabilization of dynamic unfolded states and the trapping of folding intermediates funnel the FhuA polypeptide toward the native conformation. PMID:26344570

  20. Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

    PubMed

    Booth, Laurence; Shuch, Brian; Albers, Thomas; Roberts, Jane L; Tavallai, Mehrad; Proniuk, Stefan; Zukiwski, Alexander; Wang, Dasheng; Chen, Ching-Shih; Bottaro, Don; Ecroyd, Heath; Lebedyeva, Iryna O; Dent, Paul

    2016-03-15

    We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types. PMID:26887051

  1. Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function

    PubMed Central

    Roberts, Jane L.; Tavallai, Mehrad; Proniuk, Stefan; Zukiwski, Alexander; Wang, Dasheng; Chen, Ching-Shih; Bottaro, Don; Ecroyd, Heath; Lebedyeva, Iryna O.; Dent, Paul

    2016-01-01

    We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperonechaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types. PMID:26887051

  2. Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia.

    PubMed

    Ganguly, Siddhartha; Home, Trisha; Yacoub, Abdulraheem; Kambhampati, Suman; Shi, Huidong; Dandawate, Prasad; Padhye, Subhash; Saluja, Ashok K; McGuirk, Joseph; Rao, Rekha

    2015-10-13

    CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL. PMID:26397138

  3. Tubulin-specific Chaperones: Components of a Molecular Machine that Assembles the α/β Heterodimer

    PubMed Central

    Tian, Guoling; Cowan, Nicholas J.

    2016-01-01

    The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin, CCT. There is free exchange β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCD/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin-bound (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of GDP-bound heterodimer, which becomes polymerization competent following spontaneous E-site exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a non-minimal energy state. Here we describe methods for the purification of recombinant TBC’s as biologically active proteins following their expression in a variety of host/vector systems. PMID:23973072

  4. Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE.

    PubMed

    Melero, Roberto; Moro, Fernando; Pérez-Calvo, María Ángeles; Perales-Calvo, Judit; Quintana-Gallardo, Lucía; Llorca, Oscar; Muga, Arturo; Valpuesta, José María

    2015-04-17

    Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function. PMID:25739641

  5. Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia

    PubMed Central

    Yacoub, Abdulraheem; Kambhampati, Suman; Shi, Huidong; Dandawate, Prasad; Padhye, Subhash; Saluja, Ashok K.; McGuirk, Joseph; Rao, Rekha

    2015-01-01

    CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2−/−IL2Rγc−/− mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL. PMID:26397138

  6. Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

    PubMed

    Bird, Jonathan E; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R; Friedman, Thomas B

    2014-08-26

    Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

  7. Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

    SciTech Connect

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H. Prodromou, Chrisostomos

    2015-05-01

    A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

  8. Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor*

    PubMed Central

    Bergmayr, Christian; Thurner, Patrick; Keuerleber, Simon; Kudlacek, Oliver; Nanoff, Christian; Freissmuth, Michael; Gruber, Christian W.

    2013-01-01

    The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors. PMID:23965991

  9. Periplasmic chaperone FkpA is essential for imported colicin M toxicity

    PubMed Central

    Hullmann, Julia; Patzer, Silke I; Römer, Christin; Hantke, Klaus; Braun, Volkmar

    2008-01-01

    Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 104-fold dilutions killed fkpA+ cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains. PMID:18554332

  10. Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation.

    PubMed

    Mogk, Axel; Kummer, Eva; Bukau, Bernd

    2015-01-01

    Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity. PMID:26042222

  11. Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation

    PubMed Central

    Mogk, Axel; Kummer, Eva; Bukau, Bernd

    2015-01-01

    Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity. PMID:26042222

  12. Reactivation of protein aggregates by mortalin and Tid1--the human mitochondrial Hsp70 chaperone system.

    PubMed

    Iosefson, Ohad; Sharon, Shelly; Goloubinoff, Pierre; Azem, Abdussalam

    2012-01-01

    The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria. PMID:21811887

  13. HSP33 in eukaryotes - an evolutionary tale of a chaperone adapted to photosynthetic organisms.

    PubMed

    Segal, Na'ama; Shapira, Michal

    2015-06-01

    HSP33 was originally identified in bacteria as a redox-sensitive chaperone that protects unfolded proteins from aggregation. Here, we describe a eukaryote ortholog of HSP33 from the green algae Chlamydomonas reinhardtii, which appears to play a protective role under light-induced oxidizing conditions. The algal HSP33 exhibits chaperone activity, as shown by citrate synthase aggregation assays. Studies from the Jakob laboratory established that activation of the bacterial HSP33 upon its oxidation initiates by the release of pre-bound Zn from the well conserved Zn-binding motif Cys-X-Cys-Xn -Cys-X-X-Cys, and is followed by significant structural changes (Reichmann et al., ). Unlike the bacterial protein, the HSP33 from C. reinhardtii had lost the first cysteine residue of its center, diminishing Zn-binding activity under all conditions. As a result, the algal protein can be easily activated by minor structural changes in response to oxidation and/or excess heat. An attempt to restore the missing first cysteine did not have a major effect on Zn-binding and on the mode of activation. Replacement of all remaining cysteines abolished completely any residual Zn binding, although the chaperone activation was maintained. A phylogenetic analysis of the algal HSP33 showed that it clusters with the cyanobacterial protein, in line with its biochemical localization to the chloroplast. Indeed, expression of the algal HSP33 increases in response to light-induced oxidative stress, which is experienced routinely by photosynthetic organisms. Despite the fact that no ortholog could be found in higher eukaryotes, its abundance in all algal species examined could have a biotechnological relevance. PMID:25892083

  14. Harnessing Chaperones to Generate Small-Molecule Inhibitors of Amyloid β Aggregation

    NASA Astrophysics Data System (ADS)

    Gestwicki, Jason E.; Crabtree, Gerald R.; Graef, Isabella A.

    2004-10-01

    Protein aggregation is involved in the pathogenesis of neurodegenerative diseases and hence is considered an attractive target for therapeutic intervention. However, protein-protein interactions are exceedingly difficult to inhibit. Small molecules lack sufficient steric bulk to prevent interactions between large peptide surfaces. To yield potent inhibitors of β-amyloid (Aβ) aggregation, we synthesized small molecules that increase their steric bulk by binding to chaperones but also have a moiety available for interaction with Aβ. This strategy yields potent inhibitors of Aβ aggregation and could lead to therapeutics for Alzheimer's disease and other forms of neurodegeneration.

  15. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    SciTech Connect

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  16. The Mitochondrial Chaperone TRAP1 Promotes Neoplastic Growth by Inhibiting Succinate Dehydrogenase

    PubMed Central

    Sciacovelli, Marco; Guzzo, Giulia; Morello, Virginia; Frezza, Christian; Zheng, Liang; Nannini, Nazarena; Calabrese, Fiorella; Laudiero, Gabriella; Esposito, Franca; Landriscina, Matteo; Defilippi, Paola; Bernardi, Paolo; Rasola, Andrea

    2013-01-01

    Summary We report that the mitochondrial chaperone TRAP1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. TRAP1 binds to and inhibits succinate dehydrogenase (SDH), the complex II of the respiratory chain. The respiratory downregulation elicited by TRAP1 interaction with SDH promotes tumorigenesis by priming the succinate-dependent stabilization of the proneoplastic transcription factor HIF1α independently of hypoxic conditions. These findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify TRAP1 as a promising antineoplastic target. PMID:23747254

  17. Histone deacetylase inhibitors increase glucocerebrosidase activity in Gaucher disease by modulation of molecular chaperones

    PubMed Central

    Yang, Chunzhang; Rahimpour, Shervin; Lu, Jie; Pacak, Karel; Ikejiri, Barbara; Brady, Roscoe O.; Zhuang, Zhengping

    2013-01-01

    Gaucher disease is caused by mutations of the GBA gene that encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA mutations often result in protein misfolding and premature degradation, but usually exert less effect on catalytic activity. In this study, we identified the molecular mechanism by which histone deacetylase inhibitors increase the quantity and activity of GCase. Specifically, these inhibitors limit the deacetylation of heat shock protein 90, resulting in less recognition of the mutant peptide and GCase degradation. These findings provide insight into a possible therapeutic strategy for Gaucher disease and other genetic disorders by modifying molecular chaperone and protein degradation pathways. PMID:23277556

  18. Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.

    PubMed

    Cammisa, Marco; Correra, Antonella; Andreotti, Giuseppina; Cubellis, Maria Vittoria

    2013-01-01

    Fabry_CEP is a user-friendly web-application designed to help clinicians Choose Eligible Patients for the therapy with pharmacological chaperones. It provides a database and a predictive tool to evaluate the responsiveness of lysosomal alpha-galactosidase mutants to a small molecule drug, namely 1-Deoxy-galactonojirimycin. The user can introduce any missense/nonsense mutation in the coding sequence, learn whether it is has been tested and gain access to appropriate reference literature. In the absence of experimental data structural, functional and evolutionary analysis provides a prediction and the probability that a given mutation is responsive to the drug. PMID:23883437

  19. Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli.

    PubMed

    Arié, J P; Sassoon, N; Betton, J M

    2001-01-01

    The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm. PMID:11123702

  20. A platform to view huntingtin exon 1 aggregation flux in the cell reveals divergent influences from chaperones hsp40 and hsp70.

    PubMed

    Ormsby, Angelique R; Ramdzan, Yasmin M; Mok, Yee-Foong; Jovanoski, Kristijan D; Hatters, Danny M

    2013-12-27

    Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. PMID:24196953

  1. A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70*

    PubMed Central

    Ormsby, Angelique R.; Ramdzan, Yasmin M.; Mok, Yee-Foong; Jovanoski, Kristijan D.; Hatters, Danny M.

    2013-01-01

    Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. PMID:24196953

  2. Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences.

    PubMed

    Hebert, Daniel N; Clerico, Eugenia M; Gierasch, Lila M

    2016-09-01

    In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control. PMID:27588598

  3. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-04-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  4. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

    PubMed

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ. PMID:27112153

  5. Mass spectrometric study of gas-phase ions of acid β-glucosidase (Cerezyme) and iminosugar pharmacological chaperones.

    PubMed

    Rajabi, Khadijeh

    2014-10-01

    The effect on the conformations and stability of gas-phase ions of Cerezyme, a glycoprotein, when bound to three small-molecule chaperones has been studied using intact ESI MS, collision cross section and MS/MS measurements. To distinguish between the peaks from apo and small-molecule complex ions, Cerezyme is deglycosylated (dg-Cer). ESI MS of dg-Cer reveals that glycosylation accounts for 8.5% of the molecular weight. When excess chaperone, either covalent (2FGF) or noncovalent (A and B iminosugars), is added to solutions of dg-Cer, mass spectra show peaks from 1:1 chaperone-enzyme complexes as well as free enzyme. On average, ions of the apoenzyme have 1.6 times higher cross sections when activated in the source region of the mass spectrometer. For a given charge state, ions of complexes of 2FGF and B have about 30% and 8.4% lower cross sections, respectively, compared to the apoenzyme. Thus, binding the chaperones causes the gas-phase protein to adopt more compact conformations. The noncovalent complex ions dissociate by the loss of charged chaperones. In the gas phase, the relative stability of dg-Cer with B is higher than that with the A, whereas in solution A binds enzyme more strongly than B. Nevertheless, the disagreement is explained based on the greater number of contacts between the B and dg-Cer than the A and dg-Cer (13 vs. 8), indicating the importance of noncovalent interactions within the protein-chaperone complex in the absence of solvent. Findings in this work suggest a hypothesis towards predicting a consistent correlation between gas-phase properties to solution binding properties. PMID:25303390

  6. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  7. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    PubMed Central

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  8. Diltiazem, a L-type Ca(2+) channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells.

    PubMed

    Rigat, Brigitte; Mahuran, Don

    2009-04-01

    Recently, inhibition of L-type Ca(2+) channels, using either Diltiazem or Verapamil, has been reported to partially restore mutant glucocerebrosidase activity in cells from patients with Gaucher disease homozygous for the N370S or L444P alleles, as well as cells from patients with two other lysosomal storage diseases. It was hypothesized that these drugs act on the endoplasmic reticulum, increasing its folding efficiency, inhibited due to altered calcium homeostasis. Several other laboratories have reported that cells carrying either the N370S or the F213I alleles are amenable to enzyme enhancement therapy with pharmacological chaperones, whereas cells homozygous for L444P respond poorly. We found that Verapamil treatment does not enhance mutant enzyme activity in any of the cell lines tested, while Diltiazem moderately increases activity in normal cells, and in N370S/N370S and F213I/L444P, but not in L444P/L444P Gaucher cells, or in either of two adult Tay-Sachs disease cell lines. Since the mode of action of pharmacological chaperones and Diltiazem are believed to be different, we examined the possibility that they could act in concert. Diltiazem co-administered with known chaperones failed to increase enzyme activities above that reached by chaperone-treatment alone in any of the patient cell lines. Thus, we re-examined the possibility that Diltiazem acts as a pharmacological chaperone. We found that, at the acidic pH of lysosomes, Diltiazem was not an inhibitor, nor did its presence increase the heat stability of glucocerebrosidase. However, at neutral pH, found in the endoplasmic reticulum, Diltiazem exhibited both of these properties. Thus Diltiazem exhibits the biochemical characteristics of a glucocerebrosidase pharmacological chaperone. PMID:19167257

  9. The Role of Monocarboxylate Transporters and Their Chaperone CD147 in Lactate Efflux Inhibition and the Anticancer Effects of Terminalia chebula in Neuroblastoma Cell Line N2-A

    PubMed Central

    Messeha, S. S.; Zarmouh, N. O.; Taka, E.; Gendy, S. G.; Shokry, G. R.; Kolta, M. G.; Soliman, K. F. A.

    2016-01-01

    Aims In the presence of oxygen, most of the synthesized pyruvate during glycolysis in the cancer cell of solid tumors is released away from the mitochondria to form lactate (Warburg Effect). To maintain cell homeostasis, lactate is transported across the cell membrane by monocarboxylate transporters (MCTs). The major aim of the current investigation is to identify novel compounds that inhibit lactate efflux that may lead to identifying effective targets for cancer treatment. Study Design In this study, 900 ethanol plant extracts were screened for their lactate efflux inhibition using neuroblastoma (N2-A) cell line. Additionally, we investigated the mechanism of inhibition for the most potent plant extract regarding monocarboxylate transporters expression, and consequences effects on viability, growth, and apoptosis. Methodology The potency of lactate efflux inhibition of ethanol plant extracts was evaluated in N2-A cells by measuring extracellular lactate levels. Caspase 3- activity and acridine orange/ethidium bromide staining were performed to assess the apoptotic effect. The antiproliferative effect was measured using WST assay. Western blotting was performed to quantify protein expression of MCTs and their chaperone CD147 in treated cells lysates. Results Terminalia chebula plant extract was the most potent lactate efflux inhibitor in N2-A cells among the 900 - tested plant extracts. The results obtained show that extract of Terminalia chebula fruits (TCE) significantly (P = 0.05) reduced the expression of the MCT1, MCT3, MCT4 and the chaperone CD147. The plant extract was more potent (IC50 of 3.59 ± 0.26 μg/ml) than the MCT standard inhibitor phloretin (IC50 76.54 ± 3.19 μg/ml). The extract also showed more potency and selective cytotoxicity in cancer cells than DI-TNC1 primary cell line (IC50 7.37 ± 0.28 vs. 17.35 ± 0.19 μg/ml). Moreover, TCE Inhibited N2-A cell growth (IG50 = 5.20 ± 0.30 μg/ml) and induced apoptosis at the 7.5 μg/ml concentration

  10. Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

    PubMed

    Stetz, Gabrielle; Verkhivker, Gennady M

    2016-08-22

    Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric

  11. Water and molecular chaperones act as weak links of protein folding networks: energy landscape and punctuated equilibrium changes point towards a game theory of proteins.

    PubMed

    Kovács, István A; Szalay, Máté S; Csermely, Peter

    2005-04-25

    Water molecules and molecular chaperones efficiently help the protein folding process. Here we describe their action in the context of the energy and topological networks of proteins. In energy terms water and chaperones were suggested to decrease the activation energy between various local energy minima smoothing the energy landscape, rescuing misfolded proteins from conformational traps and stabilizing their native structure. In kinetic terms water and chaperones may make the punctuated equilibrium of conformational changes less punctuated and help protein relaxation. Finally, water and chaperones may help the convergence of multiple energy landscapes during protein-macromolecule interactions. We also discuss the possibility of the introduction of protein games to narrow the multitude of the energy landscapes when a protein binds to another macromolecule. Both water and chaperones provide a diffuse set of rapidly fluctuating weak links (low affinity and low probability interactions), which allow the generalization of all these statements to a multitude of networks. PMID:15848154

  12. Chaperoned amyloid proteins for immune manipulation: α-Synuclein/Hsp70 shifts immunity toward a modulatory phenotype

    PubMed Central

    Labrador-Garrido, Adahir; Cejudo-Guillén, Marta; Klippstein, Rebecca; De Genst, Erwin J; Tomas-Gallardo, Laura; Leal, María M; Villadiego, Javier; Toledo-Aral, Juan J; Dobson, Christopher M; Pozo, David; Roodveldt, Cintia

    2014-01-01

    α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical “chaperone” activities and their recently discovered and diverse “immunoactive” properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an αSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the αSyn/Hsp70 complex, including a restrained αSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4+CD25+Foxp3+) and Teff (CD4+Foxp3−) cell populations, as opposed to significant changes in mice immunized with αSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against αSyn challenge for the “αSyn/Hsp70” experimental group as measured by IFN-γ and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-γ and immunomodulatory IL-10 indicated a unique shift toward an immunomodulatory/immunoprotective phenotype in mice immunized with the αSyn/Hsp70 complex. Overall, we propose the use of functional “HSP-chaperoned amyloid/aggregating proteins” generated with appropriate HSP-substrate protein combinations, such as the αSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or “misfolding” neurodegenerative disorders. PMID:25866630

  13. Targeting ligand-operated chaperone sigma-1 receptors in the treatment of neuropsychiatric disorders

    PubMed Central

    Teruo, Hayashi; Shang-Yi, Tsai; Tomohisa, Mori; Michiko, Fujimoto; Tsung-Ping, Su

    2011-01-01

    Introduction Current conventional therapeutic drugs for the treatment of psychiatric or neurodegenerative disorders have certain limitations of use. Psychotherapeutic drugs such as typical and atypical antipsychotics, tricyclic antidepressants, and selective monoamine reuptake inhibitors, aim to normalize the hyper- or hypo-neurotransmission of monoaminergic systems. Despite their great contribution to the outcomes of psychiatric patients, these agents often exert severe side effects and require chronic treatments to promote amelioration of symptoms. Furthermore, drugs available for the treatment of neurodegenerative disorders are severely limited. Areas covered This review discusses recent evidence that has shed light on sigma-1 receptor ligands, which may serve as a new class of antidepressants or neuroprotective agents. Sigma-1 receptors are novel ligand-operated molecular chaperones regulating a variety of signal transduction, ER stress, cellular redox, cellular survival, and synaptogenesis. Selective sigma-1 receptor ligands exert rapid antidepressant-like, anxiolytic, antinociceptive and robust neuroprotective actions in preclinical studies. The review also looks at recent studies which suggest that reactive oxygen species might play a crucial role as signal integrators at the downstream of Sig-1Rs Expert opinion The significant advances in sigma receptor research in the last decade have begun to elucidate the intracellular signal cascades upstream and downstream of sigma-1 receptors. The novel ligand-operated properties of the sigma-1 receptor chaperone may enable a variety of interventions by which stress-related cellular systems are pharmacologically controlled. PMID:21375464

  14. Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

    PubMed

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

    2015-04-14

    The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. PMID:25843722

  15. Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones.

    PubMed Central

    Gaudin, Y

    1997-01-01

    Four well-characterized monoclonal antibodies (MAbs) directed against rabies virus glycoprotein (G) were used to study G folding in vivo. Two of the MAbs were able to immunoprecipitate incompletely oxidized folding intermediates. The two others recognized G only after folding was completed. By using these MAbs, the ability of G to undergo low-pH-induced conformational changes during folding was also investigated. It appeared that some domains acquire this ability before folding is completed. In addition, interactions between unfolded G and some of the molecular chaperones were analyzed. Unfolded G was associated with BiP and calnexin. Association with BiP was maximal immediately after the pulse, whereas association with calnexin was maximal after 5 to 10 min of chase. The effects of tunicamycin and castanospermine on chaperone binding and folding were also studied. In the presence of both drugs, calnexin binding was reduced, consistent with the view that calnexin specifically recognizes monoglucosylated oligosaccharides, but some residual binding was still observed, indicating that calnexin also recognizes the polypeptide chain. In the presence of both drugs, association with BiP was increased and prolonged and folding was impaired. However, the global effects of the drugs were different, since folding was much more efficient in the presence of castanospermine than in the presence of tunicamycin. Taken together, these results provide the basis to draw a schematic view of rabies virus glycoprotein folding. PMID:9094649