Science.gov

Sample records for collagen chaperone protein

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair.

    PubMed

    Oecal, Sinan; Socher, Eileen; Uthoff, Matthias; Ernst, Corvin; Zaucke, Frank; Sticht, Heinrich; Baumann, Ulrich; Gebauer, Jan M

    2016-06-10

    Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident collagen-specific chaperone and essential for proper formation of the characteristic collagen triple helix. It preferentially binds to the folded conformation of its clients and accompanies them from the ER to the Golgi compartment, where it releases them and is recycled back to the ER. Unlike other chaperones, the binding and release cycles are not governed by nucleotide exchange and hydrolysis, but presumably the dissociation of the HSP47-procollagen complex is triggered by the lower pH in the Golgi (pH 6.3) compared with the ER (pH 7.4). Histidine residues have been suggested as triggers due to their approximate textbook pKa value of 6.1 for their side chains. We present here an extensive theoretical and experimental study of the 14 histidine residues present in canine HSP47, where we have mutated all histidine residues in the collagen binding interface and additionally all of those that were predicted to undergo a significant change in protonation state between pH 7 and 6. These mutants were characterized by biolayer interferometry for their pH-dependent binding to a collagen model. One mutant (H238N) loses binding, which can be explained by a rearrangement of the Arg(222) and Asp(385) residues, which are crucial for specific collagen recognition. Most of the other mutants were remarkably silent, but a double mutant with His(273) and His(274) exchanged for asparagines exhibits a much less pronounced pH dependence of collagen binding. This effect is mainly caused by a lower koff at the low pH values. PMID:27129216

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. [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

  1. Mussel adhesive protein provides cohesive matrix for collagen type-1α

    PubMed Central

    Martinez Rodriguez, Nadine R.; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant loadbearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m2) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  2. Mussel adhesive protein provides cohesive matrix for collagen type-1α.

    PubMed

    Martinez Rodriguez, Nadine R; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N; Waite, J Herbert

    2015-05-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA

    PubMed Central

    1994-01-01

    Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra- molecular complexes. PMID:7962074

  8. 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

  9. 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

  10. 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.

  11. Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.

    PubMed

    Krahn, Katy Nash; Bouten, Carlijn V C; van Tuijl, Sjoerd; van Zandvoort, Marc A M J; Merkx, Maarten

    2006-03-15

    Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods. PMID:16476406

  12. 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes

    PubMed Central

    Xu, Zhe; Graham, Kourtney; Foote, Molly; Liang, Fengshan; Rizkallah, Raed; Hurt, Myra; Wang, Yanchang; Wu, Yuying; Zhou, Yi

    2013-01-01

    Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. PMID:23843611

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

    PubMed

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-04-18

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  6. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling

    PubMed Central

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-01-01

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  7. Collagen-like proteins in pathogenic E. coli strains.

    PubMed

    Ghosh, Neelanjana; McKillop, Thomas J; Jowitt, Thomas A; Howard, Marjorie; Davies, Heather; Holmes, David F; Roberts, Ian S; Bella, Jordi

    2012-01-01

    The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Stabilization of collagen through bioconversion: An insight in protein-protein interaction.

    PubMed

    Usharani, Nagarajan; Jayakumar, Gladstone Christopher; Kanth, Swarna Vinodh; Rao, Jonnalagadda Raghava

    2014-08-01

    Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering. PMID:25098180

  5. 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

  6. Visualization of a radical B12 enzyme with its G-protein chaperone

    PubMed Central

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; Banerjee, Ruma; Drennan, Catherine L.

    2015-01-01

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site. PMID:25675500

  7. 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

  8. Nanointerfacial strength between non-collagenous protein and collagen fibrils in antler bone

    PubMed Central

    Hang, Fei; Gupta, Himadri S.; Barber, Asa H.

    2014-01-01

    Antler bone displays considerable toughness through the use of a complex nanofibrous structure of mineralized collagen fibrils (MCFs) bound together by non-collagenous proteins (NCPs). While the NCP regions represent a small volume fraction relative to the MCFs, significant surface area is evolved upon failure of the nanointerfaces formed at NCP–collagen fibril boundaries. The mechanical properties of nanointerfaces between the MCFs are investigated directly in this work using an in situ atomic force microscopy technique to pull out individual fibrils from the NCP. Results show that the NCP–fibril interfaces in antler bone are weak, which highlights the propensity for interface failure at the nanoscale in antler bone and extensive fibril pullout observed at antler fracture surfaces. The adhesion between fibrils and NCP is additionally suggested as being rate dependent, with increasing interfacial strength and fracture energy observed when pullout velocity decreases. PMID:24352676

  9. 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

  10. 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.

  11. The Structure and Function of Non-Collagenous Bone Proteins

    NASA Technical Reports Server (NTRS)

    Hook, Magnus; McQuillan, David J.

    1997-01-01

    The research done under the cooperative research agreement for the project titled 'The structure and function of non-collagenous bone proteins' represented the first phase of an ongoing program to define the structural and functional relationships of the principal noncollagenous proteins in bone. An ultimate goal of this research is to enable design and execution of useful pharmacological compounds that will have a beneficial effect in treatment of osteoporosis, both land-based and induced by long-duration space travel. The goals of the now complete first phase were as follows: 1. Establish and/or develop powerful recombinant protein expression systems; 2. Develop and refine isolation and purification of recombinant proteins; 3. Express wild-type non-collagenous bone proteins; 4. Express site-specific mutant proteins and domains of wild-type proteins to enhance likelihood of crystal formation for subsequent solution of structure.

  12. 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

  13. 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

  14. 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

  15. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    NASA Astrophysics Data System (ADS)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  16. Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.

    PubMed

    Aguado, Alejandra; Fernández-Higuero, José Angel; Moro, Fernando; Muga, Arturo

    2015-08-15

    The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed. PMID:26159839

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

    PubMed Central

    Li, Kelvin; Gatmaitan, Michelle; Luo, Flora; Cattin, Jerome; Nakamura, Corelle; Holmes, William E.; Angel, Thomas E.; Peters, Marion G.; Turner, Scott M.; Hellerstein, Marc K.

    2015-01-01

    Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates. PMID:25909381

  3. A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins

    PubMed Central

    Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; Santarriaga, Stephanie; Schlanger, Simon; McGlone, Cameron; Dare, Michelle; Nix, Jay C.; Scaglione, K. Matthew; Stuehr, Dennis J.; Misra, Saurav; Page, Richard C.

    2015-01-01

    Summary The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the TPR domain of CHIP in complex with the α-helical “lid” subdomain and unstructured “tail” of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting a novel mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Post-translational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a new bipartite mode of interaction between TPR domains and their binding partners. PMID:25684577

  4. Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

    PubMed

    Nakanishi, Kotaro

    2016-09-01

    RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117

  5. A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.

    PubMed

    Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; Santarriaga, Stephanie; Schlanger, Simon; McGlone, Cameron; Dare, Michelle; Nix, Jay C; Scaglione, K Matthew; Stuehr, Dennis J; Misra, Saurav; Page, Richard C

    2015-03-01

    The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners. PMID:25684577

  6. Aha1 can act as an autonomous chaperone to prevent aggregation of stressed proteins.

    PubMed

    Tripathi, Vishwadeepak; Darnauer, Stefanie; Hartwig, Nadine R; Obermann, Wolfgang M J

    2014-12-26

    Aha1 (activator of Hsp90 ATPase) stimulates the ATPase activity of the molecular chaperone Hsp90 to accelerate the conformational cycle during which client proteins attain their final shape. Thereby, Aha1 promotes effective folding of Hsp90-dependent clients such as steroid receptors and many kinases involved in cellular signaling. In our current study, we find that Aha1 plays a novel, additional role beyond regulating the Hsp90 ATP hydrolysis rate. We propose a new concept suggesting that Aha1 acts as an autonomous chaperone and associates with stress-denatured proteins to prevent them from aggregation similar to the chaperonin GroEL. Our study reveals that an N-terminal sequence of 22 amino acids, present in human but absent from yeast Aha1, is critical for this capability. However, in lieu of fostering their refolding, Aha1 allows ubiquitination of bound clients by the E3 ubiquitin ligase CHIP. Accordingly, Aha1 may promote disposal of folding defective proteins by the cellular protein quality control. PMID:25378400

  7. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. PMID:26722004

  8. Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

    PubMed Central

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R.; Liu, Yu; Powers, David L.; Kelly, Jeffery W.; Gierasch, Lila M.; Powers, Evan T.

    2015-01-01

    SUMMARY 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 vs. aggregation vs. 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

  9. 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

  10. 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

  11. Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

    NASA Astrophysics Data System (ADS)

    Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

    2013-11-01

    Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

  12. 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

  13. 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

  14. 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

  15. Dissecting the oligonucleotide binding properties of a disordered chaperone protein using surface plasmon resonance

    PubMed Central

    Baltzinger, Mireille; Sharma, Kamal Kant; Mély, Yves; Altschuh, Danièle

    2013-01-01

    We have used surface plasmon resonance to investigate the nucleic acid binding properties of the core protein of hepatitis C virus, a disordered protein believed to chaperone the genomic RNA. It was previously shown that a peptide (peptide E) corresponding to the association of two basic clusters of core enhances the annealing and the dimerization of nucleic acid fragments derived from a stem loop (SL2) in the 3′ untranslated region of the hepatitis C virus genome. However, strong aggregation of nucleic acids by core or peptide E in the excess of the latter precluded the characterization of their binding parameters up to now. By careful design of surface plasmon resonance experiments, we obtained accurate binding parameters for the interaction of peptide E with SL2-derived oligonucleotides of different lengths and sequences, in form of stem-loop, duplex or strand. Peptide E was found to bind in a salt dependent manner to all oligonucleotides assayed. Affinity data identify at least two binding modes, of which one is independent of sequence/structure, and the other is specific to the SL2 stem-loop fold. Stoichiometry data support a multi-motif binding model allowing formation of higher-order complexes. We propose that the modular binding mode demonstrated for structured RNA-binding proteins also applies to this disordered chaperone and is relevant to its activity. PMID:24030713

  16. 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

  17. Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli.

    PubMed Central

    Bergès, H; Joseph-Liauzun, E; Fayet, O

    1996-01-01

    We have studied the export of two human proteins in the course of their production in Escherichia coli. The coding sequences of the granulocyte-macrophage colony-stimulating factor and of interleukin 13 were fused to those of two synthetic signal sequences to direct the human proteins to the bacterial periplasm. We found that the total amount of protein varies with the signal peptide-cytokine combination, as does the fraction of it that is soluble in a periplasmic extract. The possibility that the major chaperone proteins such as SecB and the GroEL-GroES and DnaK-DnaJ pairs are limiting factors for the export was tested by overexpressing one or the other of these chaperones concomitantly with the heterologous protein. The GroEL-GroES chaperone pair had no effect on protein production. Overproduction of SecB or DnaK plus DnaJ resulted in a marked increase of the quantity of human proteins in the periplasmic fraction, but this increase depends on the signal peptide-heterologous protein-chaperone association involved. PMID:8572712

  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. 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

  20. 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

  1. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    PubMed Central

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  2. Reversible thermal unfolding of a yfdX protein with chaperone-like activity.

    PubMed

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  3. 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

  4. 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

  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. Gedunin inactivates the co-chaperone p23 protein causing cancer cell death by apoptosis.

    PubMed

    Patwardhan, Chaitanya A; Fauq, Abdul; Peterson, Laura B; Miller, Charles; Blagg, Brian S J; Chadli, Ahmed

    2013-03-01

    Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. PMID:23355466

  7. Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function.

    PubMed

    Chen, Gin-Fu; Sudhahar, Varadarajan; Youn, Seock-Won; Das, Archita; Cho, Jaehyung; Kamiya, Tetsuro; Urao, Norifumi; McKinney, Ronald D; Surenkhuu, Bayasgalan; Hamakubo, Takao; Iwanari, Hiroko; Li, Senlin; Christman, John W; Shantikumar, Saran; Angelini, Gianni D; Emanueli, Costanza; Ushio-Fukai, Masuko; Fukai, Tohru

    2015-01-01

    Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1(-/-) mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease. PMID:26437801

  8. Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

    PubMed Central

    Chen, Gin-Fu; Sudhahar, Varadarajan; Youn, Seock-Won; Das, Archita; Cho, Jaehyung; Kamiya, Tetsuro; Urao, Norifumi; McKinney, Ronald D.; Surenkhuu, Bayasgalan; Hamakubo, Takao; Iwanari, Hiroko; Li, Senlin; Christman, John W.; Shantikumar, Saran; Angelini, Gianni D.; Emanueli, Costanza; Ushio-Fukai, Masuko; Fukai, Tohru

    2015-01-01

    Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1−/− mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease. PMID:26437801

  9. Lifespan Control by Redox-Dependent Recruitment of Chaperones to Misfolded Proteins.

    PubMed

    Hanzén, Sarah; Vielfort, Katarina; Yang, Junsheng; Roger, Friederike; Andersson, Veronica; Zamarbide-Forés, Sara; Andersson, Rebecca; Malm, Lisa; Palais, Gael; Biteau, Benoît; Liu, Beidong; Toledano, Michel B; Molin, Mikael; Nyström, Thomas

    2016-06-30

    Caloric restriction (CR) extends the lifespan of flies, worms, and yeast by counteracting age-related oxidation of H2O2-scavenging peroxiredoxins (Prxs). Here, we show that increased dosage of the major cytosolic Prx in yeast, Tsa1, extends lifespan in an Hsp70 chaperone-dependent and CR-independent manner without increasing H2O2 scavenging or genome stability. We found that Tsa1 and Hsp70 physically interact and that hyperoxidation of Tsa1 by H2O2 is required for the recruitment of the Hsp70 chaperones and the Hsp104 disaggregase to misfolded and aggregated proteins during aging, but not heat stress. Tsa1 counteracted the accumulation of ubiquitinated aggregates during aging and the reduction of hyperoxidized Tsa1 by sulfiredoxin facilitated clearance of H2O2-generated aggregates. The data reveal a conceptually new role for H2O2 signaling in proteostasis and lifespan control and shed new light on the selective benefits endowed to eukaryotic peroxiredoxins by their reversible hyperoxidation. PMID:27264606

  10. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    PubMed Central

    Biro, Jan C

    2005-01-01

    Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle). However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base. PMID:16137324

  11. 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

  12. 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

  13. 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

  14. The Mitochondrial Chaperone Protein TRAP1 Mitigates α-Synuclein Toxicity

    PubMed Central

    Lutz, A. Kathrin; Toegel, Jane P.; Gerhardt, Ellen; Karsten, Peter; Falkenburger, Björn; Reinartz, Andrea; Winklhofer, Konstanze F.; Schulz, Jörg B.

    2012-01-01

    Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein–induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein–expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein–induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein–induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein. PMID:22319455

  15. An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

    PubMed Central

    Herschlag, D; Khosla, M; Tsuchihashi, Z; Karpel, R L

    1994-01-01

    We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions. Images PMID:8026476

  16. 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

  17. Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

    PubMed Central

    2012-01-01

    Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981

  18. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  19. Stability of silk and collagen protein materials in space.

    PubMed

    Hu, Xiao; Raja, Waseem K; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L

    2013-01-01

    Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

  20. Stability of Silk and Collagen Protein Materials in Space

    PubMed Central

    Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.

    2013-01-01

    Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

  1. 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

  2. Three reasons protein disorder analysis makes more sense in the light of collagen.

    PubMed

    Smithers, Ben; Oates, Matt E; Tompa, Peter; Gough, Julian

    2016-05-01

    We have identified that the collagen helix has the potential to be disruptive to analyses of intrinsically disordered proteins. The collagen helix is an extended fibrous structure that is both promiscuous and repetitive. Whilst its sequence is predicted to be disordered, this type of protein structure is not typically considered as intrinsic disorder. Here, we show that collagen-encoding proteins skew the distribution of exon lengths in genes. We find that previous results, demonstrating that exons encoding disordered regions are more likely to be symmetric, are due to the abundance of the collagen helix. Other related results, showing increased levels of alternative splicing in disorder-encoding exons, still hold after considering collagen-containing proteins. Aside from analyses of exons, we find that the set of proteins that contain collagen significantly alters the amino acid composition of regions predicted as disordered. We conclude that research in this area should be conducted in the light of the collagen helix. PMID:26941008

  3. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

    PubMed Central

    Munday, Diane C.; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D.; Dawson, Jenna M.; Aljabr, Waleed; Easton, Andrew J.; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M.; Ventura, Armando M.; Hughes, David J.; Barr, John N.; Fearns, Rachel; Digard, Paul

    2014-01-01

    ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is

  4. Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked

    PubMed Central

    Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche

    2013-01-01

    Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805

  5. Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.

    PubMed

    Gounel, Sébastien; Rouhana, Jad; Stines-Chaumeil, Claire; Cadet, Marine; Mano, Nicolas

    2016-07-20

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%. PMID:27165502

  6. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    PubMed Central

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  7. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    PubMed

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion. PMID:26451841

  8. 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

  9. Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

    PubMed

    Boudry, P; Gracia, C; Monot, M; Caillet, J; Saujet, L; Hajnsdorf, E; Dupuy, B; Martin-Verstraete, I; Soutourina, O

    2014-09-01

    Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria. PMID:24982306

  10. Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile

    PubMed Central

    Boudry, P.; Gracia, C.; Monot, M.; Caillet, J.; Saujet, L.; Hajnsdorf, E.; Dupuy, B.; Martin-Verstraete, I.

    2014-01-01

    Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria. PMID:24982306

  11. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    SciTech Connect

    Qualley, Dominic F. Sokolove, Victoria L.; Ross, James L.

    2015-03-13

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.

  12. 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

  13. The HSP70 co-chaperone DNAJC14 targets misfolded pendrin for unconventional protein secretion

    PubMed Central

    Jung, Jinsei; Kim, Jiyoon; Roh, Shin Hye; Jun, Ikhyun; Sampson, Robert D.; Gee, Heon Yung; Choi, Jae Young; Lee, Min Goo

    2016-01-01

    Mutations in SLC26A4, which encodes pendrin, are responsible for hearing loss with an enlarged vestibular aqueduct and Pendred syndrome. The most prevalent mutation in East Asia is p.H723R (His723Arg), which leads to defects in protein folding and cell-surface expression. Here we show that H723R-pendrin can be rescued to the cell surface by an HSP70 co-chaperone DNAJC14-dependent unconventional trafficking pathway. Blockade of ER-to-Golgi transport or activation of ER stress signals induced Golgi-independent cell-surface expression of H723R-pendrin and restored its cell-surface Cl−/HCO3− exchange activity. Proteomic and short interfering RNA screenings with subsequent molecular analyses showed that Hsc70 and DNAJC14 are required for the unconventional trafficking of H723R-pendrin. Moreover, DNAJC14 upregulation was able to induce the unconventional cell-surface expression of H723R-pendrin. These results indicate that Hsc70 and DNAJC14 play central roles in ER stress-associated unconventional protein secretion and are potential therapeutic targets for diseases such as Pendred syndrome, which arise from transport defects of misfolded proteins. PMID:27109633

  14. 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

  15. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins

    PubMed Central

    Hsiao, Tony W.; Tresco, Patrick A.; Hlady, Vladimir

    2014-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  16. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  17. 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

  18. A Review of Acquired Thermotolerance, Heat Shock Proteins, and Molecular Chaperones in Archaea: Heat Shock in Archaea

    DOE R&D Accomplishments Database

    Trent, J. D.

    1996-02-09

    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.

  19. Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis.

    PubMed

    Lo, Hui-Fen; Chen, Bo-En; Lin, Min-Guan; Chi, Meng-Chun; Wang, Tzu-Fan; Lin, Long-Liu

    2016-04-01

    Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg(2+) ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions. PMID:26743745

  20. 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

  1. Age-related changes in total protein and collagen metabolism in rat liver.

    PubMed

    Mays, P K; McAnulty, R; Laurent, G J

    1991-12-01

    Liver collagen levels are determined by a balance between synthesis and degradation, processes known to have rapid rates in growing animals. We report age-related changes in liver collagen synthesis and degradation rates, as well as protein synthesis rates, in rats at five ages from 1 to 24 mo. Fractional collagen synthesis rates were determined after injection of [14C]proline with a flooding dose of unlabeled proline and its incorporation as hydroxy-[14C]proline into proteins. Fractional protein synthesis rates were based on the uptake of [14C]proline into proteins. Fractional collagen degradation rates were calculated from the difference between collagen fractional synthesis and deposition rates. Fractional rates of collagen synthesis were similar between 1 mo (23.0% +/- 4.6%/day) and 24 mo (19.6% +/- 3.4%/day) of age. Collagen deposition into the extracellular matrix was extremely low at every age studied; therefore degradation pathways accounted for the bulk of the collagen synthesized. The mean fractional synthesis rate for the total protein pool was unaltered between 1 mo (105.0% +/- 7.2%/day) and 15 mo (89.9% +/- 6.0%/day) of age, after which it increased to 234.9% +/- 33.0%/day (p less than 0.05) by 24 mo of age. These results indicate that liver collagen and total protein synthesis rates were maintained at relatively high levels during development and maturity but that protein synthesis rates were highest in senescent animals. PMID:1959872

  2. Extracellular heat shock protein 90 binding to TGFβ receptor I participates in TGFβ-mediated collagen production in myocardial fibroblasts.

    PubMed

    García, Raquel; Merino, David; Gómez, Jenny M; Nistal, J Francisco; Hurlé, María A; Cortajarena, Aitziber L; Villar, Ana V

    2016-10-01

    The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor β (TGFβ) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling by stabilizing the TGFβ signaling cascade. We detected extracellular Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. This was supported by in silico studies predicting Hsp90 dimers and TGFβRI extracellular domain interaction. Both, Hsp90aa1 and Hsp90ab1 isoforms participate in TGFβRI complex. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFβ signaling cascade, and collagen protein synthesis was drastically reduced in Hsp90aa1 KO mice. These observations together with the significant increase in activity of Hsp90 at the plasma membrane pointed to a functional cooperative partnership between Hsp90 and TGFβRI in the fibrotic process. We propose that a surface population of Hsp90 extracellularly binds TGFβRI and this complex behaves as an active participant in collagen production in TGFβ-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 and its isoforms during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice. PMID:27418101

  3. DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

    PubMed

    Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

    2016-04-24

    DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. PMID:26975886

  4. 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

  5. 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

  6. At the Start of the Sarcomere: A Previously Unrecognized Role for Myosin Chaperones and Associated Proteins during Early Myofibrillogenesis

    PubMed Central

    Myhre, J. Layne; Pilgrim, David B.

    2012-01-01

    The development of striated muscle in vertebrates requires the assembly of contractile myofibrils, consisting of highly ordered bundles of protein filaments. Myofibril formation occurs by the stepwise addition of complex proteins, a process that is mediated by a variety of molecular chaperones and quality control factors. Most notably, myosin of the thick filament requires specialized chaperone activity during late myofibrillogenesis, including that of Hsp90 and its cofactor, Unc45b. Unc45b has been proposed to act exclusively as an adaptor molecule, stabilizing interactions between Hsp90 and myosin; however, recent discoveries in zebrafish and C. elegans suggest the possibility of an earlier role for Unc45b during myofibrillogenesis. This role may involve functional control of nonmuscle myosins during the earliest stages of myogenesis, when premyofibril scaffolds are first formed from dynamic cytoskeletal actin. This paper will outline several lines of evidence that converge to build a model for Unc45b activity during early myofibrillogenesis. PMID:22400118

  7. Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU

    PubMed Central

    Halavaty, Andrei S.; Borek, Dominika; Tyson, Gregory H.; Veesenmeyer, Jeff L.; Shuvalova, Ludmilla; Minasov, George; Otwinowski, Zbyszek

    2012-01-01

    Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin. PMID:23166655

  8. Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

    PubMed Central

    Kan, Tsung-Wai; Chalkley, Gillian E.; Sap, Karen; Bezstarosti, Karel; Demmers, Jeroen A.; Ozgur, Zeliha; van Ijcken, Wilfred F. J.; Verrijzer, C. Peter

    2013-01-01

    Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis. PMID:24086141

  9. AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ*

    PubMed Central

    Sugimoto, Shinya; Yamanaka, Kunitoshi; Nishikori, Shingo; Miyagi, Atsushi; Ando, Toshio; Ogura, Teru

    2010-01-01

    AAA+ chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers. PMID:20022957

  10. Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex

    PubMed Central

    Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J.; Rogala, Kacper B.; Deane, Charlotte M.; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G.

    2015-01-01

    Summary Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. PMID:26096973

  11. Engineering multiple biological functional motifs into a blank collagen-like protein template from Streptococcus pyogenes.

    PubMed

    Peng, Yong Y; Stoichevska, Violet; Schacht, Kristin; Werkmeister, Jerome A; Ramshaw, John A M

    2014-07-01

    Bacterially derived triple-helical, collagen-like proteins are attractive as potential biomedical materials. The collagen-like domain of the Scl2 protein from S. pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triple-helix of animal collagens. It can, therefore, be considered as a structurally-stable "blank slate" into which various defined, biological sequences, derived from animal collagens, can be added by substitutions or insertions, to enable production of novel designed materials to fit specific functional requirements. In the present study, we have used site directed mutagenesis to substitute two functional sequences, one for heparin binding and the other for integrin binding, into different locations in the triple-helical structure. This provided three new constructs, two containing the single substitutions and one containing both substitutions. The stability of these constructs was marginally reduced when compared to the unmodified sequence. When compared to the unmodified bacterial collagen, both the modified collagens that contain the heparin binding site showed marked binding of fluorescently labeled heparin. Similarly, the modified collagens from both constructs containing the integrin binding site showed significant adhesion of L929 cells that are known to possess the appropriate integrin receptor. C2C12 cells that lack any appropriate integrins did not bind. These data show that bacterial collagen-like sequences can be modified to act like natural extracellular matrix collagens by inserting one or more unique biological domains with defined function. PMID:23913780

  12. The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones

    SciTech Connect

    Norais, Cédric; Servant, Pascale; Bouthier-de-la-Tour, Claire; Coureux, Pierre-Damien; Ithurbide, Solenne; Vannier, Françoise; Guerin, Philippe P.; Dulberger, Charles L.; Satyshur, Kenneth A.; Keck, James L.; Armengaud, Jean; Cox, Michael M.; Sommer, Suzanne

    2013-02-18

    The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.

  13. Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

    PubMed Central

    Wu, Tiyun; Datta, Siddhartha A.K.; Mitra, Mithun; Gorelick, Robert J.; Rein, Alan; Levin, Judith G.

    2010-01-01

    The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing activities required for strand transfer. Surprisingly, unlike NC, with increasing concentrations, Gag proteins drastically inhibited the DNA elongation step. This result is consistent with “nucleic acid-driven multimerization” of Gag and the reported slow dissociation of Gag from bound nucleic acid, which prevent reverse transcriptase from traversing the template (“roadblock” mechanism). Our findings illustrate one reason why NC (and not Gag) has evolved as a critical cofactor in reverse transcription, a paradigm that might also extend to other retrovirus systems. PMID:20655566

  14. Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, Glaciozyma antarctica.

    PubMed

    Yusof, Nur Athirah; Hashim, Noor Haza Fazlin; Beddoe, Travis; Mahadi, Nor Muhammad; Illias, Rosli Md; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

    2016-07-01

    The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults. PMID:27154490

  15. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    PubMed

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, 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 Pr55(Gag), 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 tRNA(Lys) 3, 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

  16. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

    PubMed Central

    Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-01-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  17. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

    PubMed

    Pillet, Benjamin; García-Gómez, Juan J; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-10-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  18. 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

  19. alpha-Crystallin protein cognates in eggs of the moth, Plodia interpunctella: possible chaperones for the follicular epithelium yolk protein.

    PubMed

    Shirk, P D; Broza, R; Hemphill, M; Perera, O P

    1998-03-01

    alpha-Crystallin protein cognates were found in germ cells of the Indianmeal moth, Plodia interpunctella (Shirk and Zimowska, 1997). A cDNA clone of 674 bp with a single open reading frame was isolated for a 25,000 molecular weight polypeptide member of this family, alpha CP25, and a single transcript of approximately 700 bp was found in the ovary of vitellogenic females. Both the DNA sequence and predicted amino acid sequence showed considerable homology with the embryonic lethal gene, l(2)efl, in Drosophila melanogaster. In addition to the sequence for l(2)efl, the predicted amino acid sequence for acp25 also showed significant sequence similarly with the alpha-crystallin A chain polypeptides from the lenses of vertebrae eyes. An N-terminal hydrophobic aggregation site and a C-terminal protective binding site common to alpha-crystallin proteins were present in the predicted acp25 and l(2)efl amino acid sequences, while only the C-terminal protective binding site was present in the small heat shock protein sequences from D. melanogaster. This evidence suggests that although the alpha-crystallin protein cognates in P. interpunctella evolved from a gene common with small heat shock protein genes, the amino acid sequence has converged on a structure similar to that of alpha-crystallin proteins. Native immunoblot analysis showed that the alpha-crystallin proteins formed high molecular weight complexes with the follicular epithelium yolk protein (FEYP) but not vitellin in yolk. An electroblot binding assay was used to show that the germ-cell alpha-crystallins of P. interpunctella bind specifically with the FEYP and that the binding was reversible in the presence of ATP or low pH. This evidence in conjunction with the evidence that the alpha-crystallins and FEYP form a stable complex that co-purifies from native egg proteins suggests that the alpha-cystallin cognates function as chaperones for the follicular epithelium yolk proteins in the embryos of P. interpunctella

  20. In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

    PubMed

    Bochi, Guilherme Vargas; Torbitz, Vanessa Dorneles; de Campos, Luízi Prestes; Sangoi, Manuela Borges; Fernandes, Natieli Flores; Gomes, Patrícia; Moretto, Maria Beatriz; Barbisan, Fernanda; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

    2016-04-01

    The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2 (-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2 (-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death. PMID:26920846

  1. A rare cause of protein losing enteropathy: collagenous sprue.

    PubMed

    Karakuş, Esra; Ekinci, Özgür; Kırsaçlıoğlu, Ceyda Tuna; Özaydın, Eda; Atakan, Canan; Dursun, Ayşe

    2015-04-01

    Collagenous sprue is a clinicopathological entity with an unknown etiology. Its clinical features include progressive malabsorption, diarrhea, weight loss, unresponsiveness to treatment, and high mortality rates. The age interval of collagenous sprue is quite broad and ranges between 2 and 85 years. As far as to our knowledge, the presented case is the first reported case in infancy. PMID:25514205

  2. Binding of a Small Molecule at a Protein–Protein Interface Regulates the Chaperone Activity of Hsp70–Hsp40

    PubMed Central

    Wisén, Susanne; Bertelsen, Eric B.; Thompson, Andrea D.; Patury, Srikanth; Ung, Peter; Chang, Lyra; Evans, Christopher G.; Walter, Gladis M.; Wipf, Peter; Carlson, Heather A.; Brodsky, Jeffrey L.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.

    2010-01-01

    Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70–Hsp40 complex assembly at a native protein–protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis. PMID:20481474

  3. Group A Streptococcus Adheres to Pharyngeal Epithelial Cells with Salivary Proline-rich Proteins via GrpE Chaperone Protein*

    PubMed Central

    Murakami, Jumpei; Terao, Yutaka; Morisaki, Ichijiro; Hamada, Shigeyuki; Kawabata, Shigetada

    2012-01-01

    Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology. PMID:22566698

  4. Iron-Sulfur Cluster Biogenesis Chaperones: Evidence for Emergence of Mutational Robustness of a Highly Specific Protein-Protein Interaction.

    PubMed

    Delewski, Wojciech; Paterkiewicz, Bogumiła; Manicki, Mateusz; Schilke, Brenda; Tomiczek, Bartłomiej; Ciesielski, Szymon J; Nierzwicki, Lukasz; Czub, Jacek; Dutkiewicz, Rafal; Craig, Elizabeth A; Marszalek, Jaroslaw

    2016-03-01

    Biogenesis of iron-sulfur clusters (FeS) is a highly conserved process involving Hsp70 and J-protein chaperones. However, Hsp70 specialization differs among species. In most eukaryotes, including Schizosaccharomyces pombe, FeS biogenesis involves interaction between the J-protein Jac1 and the multifunctional Hsp70 Ssc1. But, in Saccharomyces cerevisiae and closely related species, Jac1 interacts with the specialized Hsp70 Ssq1, which emerged through duplication of SSC1. As little is known about how gene duplicates affect the robustness of their protein interaction partners, we analyzed the functional and evolutionary consequences of Ssq1 specialization on the ubiquitous J-protein cochaperone Jac1, by comparing S. cerevisiae and S. pombe. Although deletion of JAC1 is lethal in both species, alanine substitutions within the conserved His-Pro-Asp (HPD) motif, which is critical for Jac1:Hsp70 interaction, have species-specific effects. They are lethal in S. pombe, but not in S. cerevisiae. These in vivo differences correlated with in vitro biochemical measurements. Charged residues present in the J-domain of S. cerevisiae Jac1, but absent in S. pombe Jac1, are important for tolerance of S. cerevisiae Jac1 to HPD alterations. Moreover, Jac1 orthologs from species that encode Ssq1 have a higher sequence divergence. The simplest interpretation of our results is that Ssq1's coevolution with Jac1 resulted in expansion of their binding interface, thus increasing the efficiency of their interaction. Such an expansion could in turn compensate for negative effects of HPD substitutions. Thus, our results support the idea that the robustness of Jac1 emerged as consequence of its highly efficient and specific interaction with Ssq1. PMID:26545917

  5. The small heat-shock protein αB-crystallin uses different mechanisms of chaperone action to prevent the amorphous versus fibrillar aggregation of α-lactalbumin.

    PubMed

    Kulig, Melissa; Ecroyd, Heath

    2012-12-15

    Stress conditions can destabilize proteins, promoting them to unfold and adopt intermediately folded states. Partially folded protein intermediates are unstable and prone to aggregation down off-folding pathways leading to the formation of either amorphous or amyloid fibril aggregates. The sHsp (small heat-shock protein) αB-crystallin acts as a molecular chaperone to prevent both amorphous and fibrillar protein aggregation; however, the precise molecular mechanisms behind its chaperone action are incompletely understood. To investigate whether the chaperone activity of αB-crystallin is dependent upon the form of aggregation (amorphous compared with fibrillar), bovine α-lactalbumin was developed as a model target protein that could be induced to aggregate down either off-folding pathway using comparable buffer conditions. Thus when α-lactalbumin was reduced it aggregated amorphously, whereas a reduced and carboxymethylated form aggregated to form amyloid fibrils. Using this model, αB-crystallin was shown to be a more efficient chaperone against amorphously aggregating α-lactalbumin than when it aggregated to form fibrils. Moreover, αB-crystallin forms high molecular mass complexes with α-lactalbumin to prevent its amorphous aggregation, but prevents fibril formation via weak transient interactions. Thus, the conformational stability of the protein intermediate, which is a precursor to aggregation, plays a critical role in modulating the chaperone mechanism of αB-crystallin. PMID:23005341

  6. Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

    PubMed

    Belviso, Benny D; Galliani, Angela; Lasorsa, Alessia; Mirabelli, Valentina; Caliandro, Rocco; Arnesano, Fabio; Natile, Giovanni

    2016-07-01

    Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the

  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. The streptococcal collagen-binding protein CNE specifically interferes with alphaVbeta3-mediated cellular interactions with triple helical collagen.

    PubMed

    van Wieringen, Tijs; Kalamajski, Sebastian; Lidén, Asa; Bihan, Dominique; Guss, Bengt; Heinegård, Dick; Farndale, Richard W; Rubin, Kristofer

    2010-11-12

    Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α(V)β(3)-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α(V)β(3)-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α(V)β(3)-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β(1) integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α(V)β(3)-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α(V)β(3)-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation. PMID:20837478

  9. Interaction of Heat Shock Protein 90 and the Co-chaperone Cpr6 with Ura2, a Bifunctional Enzyme Required for Pyrimidine Biosynthesis*

    PubMed Central

    Zuehlke, Abbey D.; Wren, Nicholas; Tenge, Victoria; Johnson, Jill L.

    2013-01-01

    The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6-dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones. PMID:23926110

  10. Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.

    PubMed

    Moreno-del Alamo, María; Sánchez-Gorostiaga, Alicia; Serrano, Ana M; Prieto, Alicia; Cuéllar, Jorge; Martín-Benito, Jaime; Valpuesta, José M; Giraldo, Rafael

    2010-10-15

    Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits. PMID:20732327

  11. Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins*

    PubMed Central

    Nakamoto, Hitoshi; Fujita, Kensaku; Ohtaki, Aguru; Watanabe, Satoru; Narumi, Shoichi; Maruyama, Takahiro; Suenaga, Emi; Misono, Tomoko S.; Kumar, Penmetcha K. R.; Goloubinoff, Pierre; Yoshikawa, Hirofumi

    2014-01-01

    In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system. PMID:24415765

  12. Endoplasmic Reticulum Chaperone Protein GRP-78 Mediates Endocytosis of Dentin Matrix Protein 1*S⃞

    PubMed Central

    Ravindran, Sriram; Narayanan, Karthikeyan; Eapen, Asha Sarah; Hao, Jianjun; Ramachandran, Amsaveni; Blond, Sylvie; George, Anne

    2008-01-01

    Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant KD = 85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development. PMID:18757373

  13. 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

  14. 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

  15. Tumor matrix protein collagen XIα1 in cancer

    PubMed Central

    Raglow, Zoe; Thomas, Sufi M

    2015-01-01

    The extracellular matrix is increasingly recognized as an essential player in cancer development and progression. Collagens are one of the most important components of the extracellular matrix, and have themselves been implicated in many aspects of neoplastic transformation. Collagen XI is a minor collagen whose main physiologic function is to regulate the diameter of major collagen fibrils. The α1 chain of collagen XI (colXIα1), has known pathogenic roles in several musculoskeletal disorders. Recent research has highlighted the importance of colXIα1 in many types of cancer, including its roles in metastasis, angiogenesis, and drug resistance, as well as its potential utility in screening tests and as a therapeutic target. High levels of colXIα1 overexpression have been reported in multiple expression profile studies examining differences between cancerous and normal tissue, and between beginning and advanced stage cancer. Its expression has been linked to poor progression-free and overall survival. The consistency of this data across cancer types is particularly striking, including colorectal, ovarian, breast, head and neck, lung, and brain cancers. This review discusses the role of collagen XIα1 in cancer and its potential as a target for cancer therapy. PMID:25511741

  16. Purification of collagen-binding proteins of Lactobacillus reuteri NCIB 11951.

    PubMed

    Aleljung, P; Shen, W; Rozalska, B; Hellman, U; Ljungh, A; Wadström, T

    1994-04-01

    Collagen type-I-binding proteins of Lactobacillus reuteri NCIB 11951 were purified. The cell surface proteins were affinity purified on collagen Sepharose and eluted with an NaCl gradient. Two protein bands were eluted from the column (29 kDa and 31 kDa), and both bound radio-labeled collagen type I. Rabbit antisera raised against the 29 kDa and 31 kDa protein reacted with the affinity-purified proteins in a Western blot with whole-cell extract used as antigen. The N-terminal sequence of the 29-kDa and 31-kDa proteins demonstrated the closest homologies with internal sequences from an Escherichia coli trigger factor protein (TIG.ECOLI). Out of nine other lactobacilli, the antisera reacted only with the L. reuteri and not with the other species tested. PMID:7764651

  17. Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

    PubMed

    MacBeath, J R; Shackleton, D R; Hulmes, D J

    1993-09-15

    Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix

  18. Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import

    PubMed Central

    Bhangoo, Melanie K.; Tzankov, Stefan; Fan, Anna C.Y.; Dejgaard, Kurt; Thomas, David Y.

    2007-01-01

    Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90–preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone–preprotein complexes. PMID:17596514

  19. Link-proteins and non-collagenous proteins from normal and chondrodysplastic cartilages.

    PubMed

    Chaminade, F; Stanescu, V; Stanescu, R; Maroteaux, P

    1979-08-01

    Baboon and human articular and growth cartilage was extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea pH 6.8 the proteins were separated from proteoglycans by ion-exchange chromatography. The concentrated and reduced protein fractions was analyzed by SDS-PAGE. Bands corresponding to collagen and to 6 major non-collegenous proteins were found. Two of the latter were identified with the link-proteins. By using small columns and microconcentration procedures, a gel-electrophoretic analysis of link-proteins extracted from small pieces of cartilage was performed and ten cases of osteochondrodysplasias were studied. No abnormalities were detected in the following syndromes: achondroplasia, diastrophic dwarfism, thanatophoric dwarfism, Jeune disease, spondyloepiphyseal dysplasia congenita, Kozlowski syndrome, osteogenesis imperfecta, polyepiphyseal dysplasia with diabetes mellitus. PMID:113215

  20. 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

  1. Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions*

    PubMed Central

    Abbonante, Vittorio; Gruppi, Cristian; Rubel, Diana; Gross, Oliver; Moratti, Remigio; Balduini, Alessandra

    2013-01-01

    Growing evidence demonstrates that extracellular matrices regulate many aspects of megakaryocyte (MK) development; however, among the different extracellular matrix receptors, integrin α2β1 and glycoprotein VI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor discoidin domain receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and spleen tyrosine kinase (Syk). Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, glycoprotein VI, α2β1, and LAIR-1, determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs, and we point out that an increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment. PMID:23530036

  2. Gedunin Inactivates the Co-chaperone p23 Protein Causing Cancer Cell Death by Apoptosis*♦

    PubMed Central

    Patwardhan, Chaitanya A.; Fauq, Abdul; Peterson, Laura B.; Miller, Charles; Blagg, Brian S. J.; Chadli, Ahmed

    2013-01-01

    Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. PMID:23355466

  3. Biosynthesis of collagen and other matrix proteins by articular cartilage in experimental osteoarthrosis.

    PubMed Central

    Eyre, D R; McDevitt, C A; Billingham, M E; Muir, H

    1980-01-01

    Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage. Images Fig. 3. PMID:7470037

  4. Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans.

    PubMed

    Nomura, R; Nakano, K; Naka, S; Nemoto, H; Masuda, K; Lapirattanakul, J; Alaluusua, S; Matsumoto, M; Kawabata, S; Ooshima, T

    2012-08-01

    Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains. PMID:22759315

  5. Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells.

    PubMed

    You, Bang-Jau; Wu, Yang-Chang; Wu, Chi-Yu; Bao, Bo-Ying; Chen, Mei-Yu; Chang, Yu-Hao; Lee, Hong-Zin

    2011-08-01

    Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 μg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells. PMID:21569101

  6. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  7. Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates

    SciTech Connect

    Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlinoi, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; San Antonio, James D.

    2008-07-18

    Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The 'cell interaction domain' is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The 'matrix interaction domain' may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

  8. Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

    SciTech Connect

    Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E.

    2010-02-11

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e{sub 1} bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  9. 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

  10. Identification of protein-protein interactions between the TatB and TatC subunits of the twin-arginine translocase system and respiratory enzyme specific chaperones.

    PubMed

    Kuzniatsova, Lalita; Winstone, Tara M L; Turner, Raymond J

    2016-04-01

    The Twin-arginine translocation (Tat) pathway serves for translocation of fully folded proteins across the cytoplasmic membrane in bacterial and chloroplast thylakoid membranes. The Escherichia coli Tat system consists of three core components: TatA, TatB, and TatC. The TatB and TatC subunits form the receptor complex for Tat dependent proteins. The TatB protein is composed of a single transmembrane helix and cytoplasmic domain. The structure of TatC revealed six transmembrane helices. Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat dependent respiratory enzymes. Here we applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. We focused on the NarJ subfamily, where DmsD--the REMP for dimethyl sulfoxide reductase in E. coli--was previously shown to interact with TatB and TatC. We found that these REMPs interact with TatC cytoplasmic loops 1, 2 and 4, with the exception of NarJ, that only interacts with 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. The results demonstrate a role of REMP chaperones in targeting respiratory enzymes to the Tat system. The data suggests that the different REMPs may have different mechanisms for this task. PMID:26826271

  11. 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

  12. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

    SciTech Connect

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.

    2015-02-13

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.

  13. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

    DOE PAGESBeta

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.

    2015-02-13

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less

  14. Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

    PubMed Central

    Stetz, Gabrielle; Verkhivker, Gennady M.

    2015-01-01

    Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations

  15. 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

  16. Biophysical and biological characterisation of collagen/resilin-like protein composite fibres.

    PubMed

    Sanami, M; Shtein, Z; Sweeney, I; Sorushanova, A; Rivkin, A; Miraftab, M; Shoseyov, O; O'Dowd, C; Mullen, A M; Pandit, A; Zeugolis, D I

    2015-12-01

    Collagen type I, in various physical forms, is widely used in tissue engineering and regenerative medicine. To control the mechanical properties and biodegradability of collagen-based devices, exogenous cross-links are introduced into the 3D supramolecular structure. However, potent cross-linking methods are associated with cytotoxicity, whilst mild cross-linking methods are associated with suboptimal mechanical resilience. Herein, we assessed the influence of resilin, a super-elastic and highly stretchable protein found within structures in arthropods where energy storage and long-range elasticity are needed, on the biophysical and biological properties of mildly cross-linked extruded collagen fibres. The addition of resilin-like protein in the 4-arm poly(ethylene glycol) ether tetrasuccinimidyl glutarate cross-linked collagen fibres resulted in a significant increase of stress and strain at break values and a significant decrease of modulus values. The addition of resilin-like protein did not compromise cell metabolic activity and DNA concentration. All groups are supported parallel to the longitudinal fibre axis cell orientation. Herein we provide evidence that the addition of resilin-like protein in mildly cross-linked collagen fibres improves their biomechanical properties, without jeopardising their biological properties. PMID:26541078

  17. Collagen-binding proteins of rat mammary tumor epithelial cells: A biochemical and immunological study

    SciTech Connect

    Wirl, G.; Pfaeffle, M. )

    1988-05-01

    Collagen-binding proteins were studied in mammary epithelial cells of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Using affinity chromatography on type I collagen-Sepharose and polyacrylamide slab gel electrophoresis, three major proteins of 34,000, 36,000, and 38,000 Da were found. Pulse-chase experiments did not indicate a precursor-product relationship of these proteins. Tryptic/chymotryptic peptide maps, however, revealed that the 36,000- and 38,000-Da proteins are very similar but are quite different from the 34,000-Da molecular form. The distribution and function of these proteins were then analyzed by using polyclonal antibodies directed against the entire set of major proteins. In immunofluorescence studies the authors observed a dense, punctate distribution of fluorescence on the cell surface of isolated and unfixed epithelial organoids and a bright pericellular staining in cultures after fixation. Treatment with the antiserum did not affect attachment and spreading of cuboidal mammary cells to plastic or to a collagen substratum. However, when the antiserum was added to the medium of growing cuboidal cells, it caused the formation of duct-like structures. These studies indicate that collagen-binding proteins may play a role in mammary gland morphology.

  18. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise.

    PubMed

    Miller, Benjamin F; Olesen, Jens L; Hansen, Mette; Døssing, Simon; Crameri, Regina M; Welling, Rasmus J; Langberg, Henning; Flyvbjerg, Allan; Kjaer, Michael; Babraj, John A; Smith, Kenneth; Rennie, Michael J

    2005-09-15

    We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload (W(max)). To label tissue proteins in muscle and tendon primed, constant infusions of [1-(13)C]leucine or [1-(13)C]valine and flooding doses of [(15)N] or [(13)C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography-mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation. PMID:16002437

  19. Identification of Surface Proteins from Lactobacillus casei BL23 Able to Bind Fibronectin and Collagen.

    PubMed

    Muñoz-Provencio, Diego; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-03-01

    Strains of lactobacilli show the capacity to attach to extracellular matrix proteins. Cell-wall fractions of Lactobacillus casei BL23 enriched in fibronectin, and collagen-binding proteins were isolated. Mass spectrometry analysis of their protein content revealed the presence of stress-related proteins (GroEL, ClpL), translational elongation factors (EF-Tu, EF-G), oligopeptide solute-binding proteins, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The latter two enzymes were expressed in Escherichia coli and purified as glutathione-S-transferase (GST) fusion proteins, and their in vitro binding activity to fibronectin and collagen was confirmed. These results reinforce the idea that lactobacilli display on their surfaces a variety of moonlighting proteins that can be important in their adaptation to survive at intestinal mucosal sites and in the interaction with host cells. PMID:26781495

  20. The p23 molecular chaperone and GCN5 acetylase jointly modulate protein-DNA dynamics and open chromatin status

    PubMed Central

    Zelin, Elena; Zhang, Yang; Toogun, Oyetunji A.; Zhong, Sheng; Freeman, Brian C.

    2012-01-01

    SUMMARY Cellular processes function through multi-step pathways that are reliant on the controlled association and disassociation of sequential protein complexes. While dynamic action is critical to propagate and terminate work, the mechanisms used to disassemble biological structures are not fully understood. Here, we show that the p23 molecular chaperone initiates disassembly of protein-DNA complexes and that the GCN5 acetyltransferase prolongs the dissociated state through lysine acetylation. By modulating the DNA bound-state, we found that the conserved and essential joint activities of p23 and GCN5 impacted transcription factor activation potential and response time to an environmental cue. Notably, p23 and GCN5 were required to maintain open chromatin regions along the genome indicating that dynamic protein behavior is a critical feature of various DNA-associated events. Our data support a model in which p23 and GCN5 regulate diverse multi-step pathways by controlling the longevity of protein-DNA complexes. PMID:23022381

  1. ATP-Dependent Chromatin Remodeling by Cockayne Syndrome Protein B and NAP1-Like Histone Chaperones Is Required for Efficient Transcription-Coupled DNA Repair

    PubMed Central

    Lake, Robert J.; Basheer, Asjad; Fan, Hua-Ying

    2013-01-01

    The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome—a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP–dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP–dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB–binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP–dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome. PMID:23637612

  2. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  3. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  4. Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes

    PubMed Central

    Nano, Nardin; Houry, Walid A.

    2013-01-01

    Rvb1 and Rvb2 are highly conserved and essential eukaryotic AAA+ proteins linked to a wide range of cellular processes. AAA+ proteins are ATPases associated with diverse cellular activities and are characterized by the presence of one or more AAA+ domains. These domains have the canonical Walker A and Walker B nucleotide binding and hydrolysis motifs. Rvb1 and Rvb2 have been found to be part of critical cellular complexes: the histone acetyltransferase Tip60 complex, chromatin remodelling complexes Ino80 and SWR-C, and the telomerase complex. In addition, Rvb1 and Rvb2 are components of the R2TP complex that was identified by our group and was determined to be involved in the maturation of box C/D small nucleolar ribonucleoprotein (snoRNP) complexes. Furthermore, the Rvbs have been associated with mitotic spindle assembly, as well as phosphatidylinositol 3-kinase-related protein kinase (PIKK) signalling. This review sheds light on the potential role of the Rvbs as chaperones in the assembly and remodelling of these critical complexes. PMID:23530256

  5. 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

  6. 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

  7. Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)*

    PubMed Central

    Dutta, Dipanjan; Chattopadhyay, Shiladitya; Bagchi, Parikshit; Halder, Umesh Chandra; Nandi, Satabdi; Mukherjee, Anupam; Kobayashi, Nobumichi; Taniguchi, Koki; Chawla-Sarkar, Mamta

    2011-01-01

    Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225–258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225–258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225–258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225–258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells. PMID:21489987

  8. Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity

    PubMed Central

    Ishibashi, Nanako; Kitakura, Saeko; Terakura, Shinji; Machida, Chiyoko; Machida, Yasunori

    2014-01-01

    Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants. PMID:25389429

  9. The cleverSuite approach for protein characterization: predictions of structural properties, solubility, chaperone requirements and RNA-binding abilities

    PubMed Central

    Klus, Petr; Bolognesi, Benedetta; Agostini, Federico; Marchese, Domenica; Zanzoni, Andreas; Tartaglia, Gian Gaetano

    2014-01-01

    Motivation: The recent shift towards high-throughput screening is posing new challenges for the interpretation of experimental results. Here we propose the cleverSuite approach for large-scale characterization of protein groups. Description: The central part of the cleverSuite is the cleverMachine (CM), an algorithm that performs statistics on protein sequences by comparing their physico-chemical propensities. The second element is called cleverClassifier and builds on top of the models generated by the CM to allow classification of new datasets. Results: We applied the cleverSuite to predict secondary structure properties, solubility, chaperone requirements and RNA-binding abilities. Using cross-validation and independent datasets, the cleverSuite reproduces experimental findings with great accuracy and provides models that can be used for future investigations. Availability: The intuitive interface for dataset exploration, analysis and prediction is available at http://s.tartaglialab.com/clever_suite. Contact: gian.tartaglia@crg.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24493033

  10. LcBiP, a endoplasmic reticulum chaperone binding protein gene from Lycium chinense, confers cadmium tolerance in transgenic tobacco.

    PubMed

    Guan, Chunfeng; Jin, Chao; Ji, Jing; Wang, Gang; Li, Xiaozhou

    2015-01-01

    Cadmium (Cd) accumulation is very toxic to plants. The presence of Cd may lead to excessive production of reactive oxygen species (ROS), and then cause inhibition of plant growth. The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which has been shown to function as a sensor of alterations in the ER environment. BiP overexpression in plants was shown to increase drought tolerance through inhibition of ROS accumulation. Due to the above relationships, it is likely that there may be a link between Cd stress tolerance, ROS accumulation and the BiP transcript expression in plants. In this study, a BiP gene, LcBiP, from L. chinense was isolated and characterized. Overexpression of LcBiP in tobacco conferred Cd tolerance. Under Cd stress conditions, the transgenic tobacco lines exhibited better chlorophyll retention, less accumulation of ROS, longer root length, more glutathione (GSH) content, and less antioxidant enzyme activity than the wild type. These data demonstrated that LcBiP act as a positive regulator in Cd stress tolerance. It is hypothesized that the improved Cd tolerance of the transgenic tobacco plants may be due to the enhanced ROS scavenging capacity. The enhancement of GSH content might contribute to this ROS scavenging capacity in the transgenic plants. However, the underlying mechanism for BiP-mediated increase in Cd stress tolerance need to be further clarified. PMID:25589446

  11. The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

    PubMed Central

    Yen, Edwin H. K.; Sodek, Jaro; Melcher, Antony H.

    1979-01-01

    Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO2 (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [3H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO2 [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [3H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO2 [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO2 for the first 48h were unable to respond to a subsequent increase in pO2 during the last 24h. Analysis of pepsin-solubilized collagen α-chains labelled with [14C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO2. Type-III collagen comprised 20–30% of the radioactivity in α-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO2 was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo. PMID:454369

  12. Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice.

    PubMed

    Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L; Jerome, Jacob A; Madsen, Daniel H; Christofidou-Solomidou, Melpo; Speicher, David W; Bachovchin, William W; Feghali-Bostwick, Carol; Puré, Ellen

    2016-04-01

    Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. PMID:26663085

  13. Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.

    PubMed

    Lamandé, S R; Shields, K A; Kornberg, A J; Shield, L K; Bateman, J F

    1999-07-30

    Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy. PMID:10419498

  14. High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

    PubMed

    Morshauser, R C; Hu, W; Wang, H; Pang, Y; Flynn, G C; Zuiderweg, E R

    1999-06-25

    The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions. PMID:10373374

  15. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

    PubMed

    Kumar, C Sudheer; Swamy, Musti J

    2016-07-01

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch. PMID:27292547

  16. A novel component of epidermal cell-matrix and cell-cell contacts: transmembrane protein type XIII collagen.

    PubMed

    Peltonen, S; Hentula, M; Hägg, P; Ylä-Outinen, H; Tuukkanen, J; Lakkakorpi, J; Rehn, M; Pihlajaniemi, T; Peltonen, J

    1999-10-01

    Type XIII collagen is a short chain collagen which has recently been shown to be a transmembrane protein. The purpose of this study was to elucidate the presence and localization of type XIII collagen in normal human skin and cultured keratinocytes. Expression of type XIII collagen was demonstrated in normal human skin and epidermis at the RNA level using reverse transcription followed by polymerase chain reaction and at the protein level using western blotting and indirect immunofluorescence labeling. Immunolabeling of epidermis revealed type XIII collagen both in the cell-cell contact sites and in the dermal-epidermal junction. In cultured keratinocytes type XIII collagen epitopes were detected in focal contacts and in intercellular contacts. The results of this study show very little colocalization of type XIII collagen and desmosomal components at the light microscopic level. Thus, these results suggest that type XIII collagen is unlikely to be a component of desmosomes. Instead, the punctate labeling pattern of type XIII collagen at the cell-cell contact sites and high degree of colocalization with E-cadherin suggests that type XIII collagen is very likely to be closely associated with adherens type junctions, and may, in fact, be a component of these junctions. PMID:10504453

  17. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    SciTech Connect

    Lee, E.Y.H.; Parry, G.; Bissell, M.J.

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce ..gamma..-casein, a member of the casein gene family, only if they are maintained on floating collagen gels. In this paper we show: (a) Cells on floating collagen gels secrete not only ..gamma..-casein but also ..cap alpha../sub 1/-, ..cap alpha../sub 2/-, and ..beta..-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane, are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

  18. 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

  19. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. PMID:26259880

  20. 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

  1. The Cytoplasmic Hsp70 Chaperone Machinery Subjects Misfolded and Endoplasmic Reticulum Import-incompetent Proteins to Degradation via the Ubiquitin–Proteasome System

    PubMed Central

    Park, Sae-Hun; Bolender, Natalia; Eisele, Frederik; Kostova, Zlatka; Takeuchi, Junko; Coffino, Philip

    2007-01-01

    The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import–defective mutated derivatives of carboxypeptidase yscY (ΔssCPY* and ΔssCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (ΔssCPY). All these protein species are rapidly degraded via the ubiquitin–proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssa1p function. Fusion of ΔssCPY* to GFP-cODC to form ΔssCPY*-GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation. PMID:17065559

  2. Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)*

    PubMed Central

    Chang, Lyra; Thompson, Andrea D.; Ung, Peter; Carlson, Heather A.; Gestwicki, Jason E.

    2010-01-01

    The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/μg/min−1. We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of ΔdnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding. PMID:20439464

  3. 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

  4. Mutations of the molecular chaperone protein SecB which alter the interaction between SecB and maltose-binding protein.

    PubMed

    Gannon, P M; Kumamoto, C A

    1993-01-25

    SecB is a 16-kDa cytosolic chaperone protein that is required for efficient export of particular proteins in Escherichia coli. To identify regions of SecB that contribute to efficient protein export, we isolated secB point mutants that are defective for protein export in vivo. We obtained missense mutations at residues Leu75 (SecBL75Q), Cys76 (SecBC76Y), and Glu77 (SecBE77K) in the center of the secB gene. In vivo, mutant SecBL75Q and SecBE77K proteins are capable of binding to precursor maltose-binding protein (MBP) and preventing the formation of export-incompetent precursor MBP; however, export of MBP is still defective. In vitro, purified SecBL75Q and SecBE77K proteins bound to unfolded MBP and blocked its refolding. SecBL75Q and SecBE77K were more effective than wild-type SecB at blocking the refolding of unfolded MBP, suggesting that SecBL75Q and SecBE77K have a higher affinity for unfolded MBP. PMID:8420934

  5. Ribosomal P3 protein AtP3B of Arabidopsis acts as both protein and RNA chaperone to increase tolerance of heat and cold stresses.

    PubMed

    Kang, Chang Ho; Lee, Young Mee; Park, Joung Hun; Nawkar, Ganesh M; Oh, Hun Taek; Kim, Min Gab; Lee, Soo In; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2016-07-01

    The P3 proteins are plant-specific ribosomal P-proteins; however, their molecular functions have not been characterized. In a screen for components of heat-stable high-molecular weight (HMW) complexes, we isolated the P3 protein AtP3B from heat-treated Arabidopsis suspension cultures. By size-exclusion chromatography (SEC), SDS-PAGE and native PAGE followed by immunoblotting with anti-AtP3B antibody, we showed that AtP3B was stably retained in HMW complexes following heat shock. The level of AtP3B mRNA increased in response to both high- and low-temperature stresses. Bacterially expressed recombinant AtP3B protein exhibited both protein and RNA chaperone activities. Knockdown of AtP3B by RNAi made plants sensitive to both high- and low-temperature stresses, whereas overexpression of AtP3B increased tolerance of both conditions. Together, our results suggest that AtP3B protects cells against both high- and low-temperature stresses. These findings provide novel insight into the molecular functions and in vivo roles of acidic ribosomal P-proteins, thereby expanding our knowledge of the protein production machinery. PMID:27004478

  6. Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli▿†

    PubMed Central

    Prasad, Shivcharan; Khadatare, Prashant B.; Roy, Ipsita

    2011-01-01

    The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium. PMID:21551288

  7. Endoplasmic reticulum stress inhibits collagen synthesis independent of collagen-modifying enzymes in different chondrocyte populations and dermal fibroblasts.

    PubMed

    Vonk, Lucienne A; Doulabi, Behrouz Zandieh; Huang, Chun-Ling; Helder, Marco N; Everts, Vincent; Bank, Ruud A

    2010-06-01

    Chondrocytes respond to glucose deprivation with a decreased collagen synthesis due to disruption of a proper functioning of the endoplasmic reticulum (ER): ER stress. Since the mechanisms involved in the decreased synthesis are unknown, we have investigated whether chaperones and collagen-modifying enzymes are affected by glucose deprivation. Chondrocytes obtained from nucleus pulposus, annulus fibrosus, articular cartilage, and meniscus and dermal fibroblasts were cultured under control conditions or exposed to the ER stress-inducing treatments of tunicamycin addition or glucose withdrawal. Both treatments resulted in an up-regulation of the gene expression of the ER stress markers in all cell types, but dermal fibroblasts showed a delayed response to glucose deprivation. Collagen gene expression was down-regulated, and less collagen protein was present in the cells under both ER stress-inducing conditions. The expression levels of the prolyl 4-hydroxylases were either not affected (P4ha3) or increased (P4ha1 and P4ha2), the levels of the lysyl hydroxylases decreased, and the N-propeptidase Adamts2 decreased. Both treatments induced apoptosis. Chondrocytes respond more quickly to glucose deprivation, but it appears that chondrocytes can cope better with tunicamycin-induced ER stress than fibroblasts. Although collagen synthesis was inhibited by the treatments, some collagen-modifying enzymes and chaperones were up-regulated, suggesting that there is no causal relation between them. PMID:20555395

  8. Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity*

    PubMed Central

    Xu, Xinping; Sarbeng, Evans Boateng; Vorvis, Christina; Kumar, Divya Prasanna; Zhou, Lei; Liu, Qinglian

    2012-01-01

    The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s. PMID:22157767

  9. 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...

  10. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis

    PubMed Central

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-01-01

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor. DOI: http://dx.doi.org/10.7554/eLife.06587.001 PMID:26052747

  11. Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP*

    PubMed Central

    Zhang, Yi; Liu, Ren; Ni, Min; Gill, Parkash; Lee, Amy S.

    2010-01-01

    The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr648 of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented. PMID:20208072

  12. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

    PubMed

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-01-01

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor. PMID:26052747

  13. Structural Features and Chaperone Activity of the NudC Protein Family

    SciTech Connect

    Zheng, Meiying; Cierpicki, Tomasz; Burdette, Alexander J.; Utepbergenov, Darkhan; Janczyk, Pawe; #322; #321; .; Derewenda, Urszula; Stukenberg, P. Todd; Caldwell, Kim A.; Derewenda, Zygmunt S.

    2012-05-25

    The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.

  14. 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

  15. 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

  16. Human Mitochondrial Chaperone (mtHSP70) and Cysteine Desulfurase (NFS1) Bind Preferentially to the Disordered Conformation, Whereas Co-chaperone (HSC20) Binds to the Structured Conformation of the Iron-Sulfur Cluster Scaffold Protein (ISCU)*

    PubMed Central

    Cai, Kai; Frederick, Ronnie O.; Kim, Jin Hae; Reinen, Nichole M.; Tonelli, Marco; Markley, John L.

    2013-01-01

    Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU. PMID:23940031

  17. The Structure and Function of Non-Collagenous Bone Proteins

    NASA Technical Reports Server (NTRS)

    Hook, Magnus

    1997-01-01

    The long-term goal for this program is to determine the structural and functional relationships of bone proteins and proteins that interact with bone. This information will used to design useful pharmacological compounds that will have a beneficial effect in osteoporotic patients and in the osteoporotic-like effects experienced on long duration space missions. The first phase of this program, funded under a cooperative research agreement with NASA through the Texas Medical Center, aimed to develop powerful recombinant expression systems and purification methods for production of large amounts of target proteins. Proteins expressed in sufficient'amount and purity would be characterized by a variety of structural methods, and made available for crystallization studies. In order to increase the likelihood of crystallization and subsequent high resolution solution of structures, we undertook to develop expression of normal and mutant forms of proteins by bacterial and mammalian cells. In addition to the main goals of this program, we would also be able to provide reagents for other related studies, including development of anti-fibrotic and anti-metastatic therapeutics.

  18. 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.

  19. 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

  20. RFX family proteins differentially interact with HDACs to repress collagen alpha 2(I) gene (COL1A2) expression

    PubMed Central

    Xu, Y.; Sengupta, P.K.; Seto, E.; Smith, B.D.

    2006-01-01

    Our studies indicate that regulatory factor for X-box (RFX) family proteins repress collagen alpha2(I) gene (COL1A2) expression (1,2). In the present investigation, we examine the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B) whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity while HDAC1 enhances inhibition of collagen promoter activity by RFX1. IFN-γ promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation (ChIP) assay. RFX1 binds to methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (aza-dC), that inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in ChIP assays. Finally, both RFX1 and RFX5 are acetylated in vivo. TSA stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression: one for RFX5/HDAC2 in IFN-γ mediated repression, the other for RFX1/HDAC1 in methylation mediated collagen silencing. PMID:16464847

  1. 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

  2. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  3. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of {approx}5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in ffolding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to

  4. Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I.

    PubMed

    Nallapareddy, S R; Qin, X; Weinstock, G M; Höök, M; Murray, B E

    2000-09-01

    Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I. PMID:10948147

  5. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    PubMed Central

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  6. Universal Stress Protein Exhibits a Redox-Dependent Chaperone Function in Arabidopsis and Enhances Plant Tolerance to Heat Shock and Oxidative Stress

    PubMed Central

    Jung, Young Jun; Melencion, Sarah Mae Boyles; Lee, Eun Seon; Park, Joung Hun; Alinapon, Cresilda Vergara; Oh, Hun Taek; Yun, Dae-Jin; Chi, Yong Hun; Lee, Sang Yeol

    2015-01-01

    Although a wide range of physiological information on Universal Stress Proteins (USPs) is available from many organisms, their biochemical, and molecular functions remain unidentified. The biochemical function of AtUSP (At3g53990) from Arabidopsis thaliana was therefore investigated. Plants over-expressing AtUSP showed a strong resistance to heat shock and oxidative stress, compared with wild-type and Atusp knock-out plants, confirming the crucial role of AtUSP in stress tolerance. AtUSP was present in a variety of structures including monomers, dimers, trimers, and oligomeric complexes, and switched in response to external stresses from low molecular weight (LMW) species to high molecular weight (HMW) complexes. AtUSP exhibited a strong chaperone function under stress conditions in particular, and this activity was significantly increased by heat treatment. Chaperone activity of AtUSP was critically regulated by the redox status of cells and accompanied by structural changes to the protein. Over-expression of AtUSP conferred a strong tolerance to heat shock and oxidative stress upon Arabidopsis, primarily via its chaperone function. PMID:26734042

  7. [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

  8. Determinants of rodent longevity in the chaperone-protein degradation network.

    PubMed

    Rodriguez, Karl A; Valentine, Joseph M; Kramer, David A; Gelfond, Jonathan A; Kristan, Deborah M; Nevo, Eviatar; Buffenstein, Rochelle

    2016-05-01

    Proteostasis is an integral component of healthy aging, ensuring maintenance of protein structural and functional integrity with concomitant impact upon health span and longevity. In most metazoans, increasing age is accompanied by a decline in protein quality control resulting in the accrual of damaged, self-aggregating cytotoxic proteins. A notable exception to this trend is observed in the longest-lived rodent, the naked mole-rat (NMR, Heterocephalus glaber) which maintains proteostasis and proteasome-mediated degradation and autophagy during aging. We hypothesized that high levels of the proteolytic degradation may enable better maintenance of proteostasis during aging contributing to enhanced species maximum lifespan potential (MLSP). We test this by examining proteasome activity, proteasome-related HSPs, the heat-shock factor 1 (HSF1) transcription factor, and several markers of autophagy in the liver and quadriceps muscles of eight rodent species with divergent MLSP. All subterranean-dwelling species had higher levels of proteasome activity and autophagy, possibly linked to having to dig in soils rich in heavy metals and where underground atmospheres have reduced oxygen availability. Even after correcting for phylogenetic relatedness, a significant (p < 0.02) positive correlation between MLSP, HSP25, HSF1, proteasome activity, and autophagy-related protein 12 (ATG12) was observed, suggesting that the proteolytic degradation machinery and maintenance of protein quality play a pivotal role in species longevity among rodents. PMID:26894765

  9. The Box H/ACA snoRNP assembly factor Shq1p is a chaperone protein homologous to Hsp90 co-chaperones that binds to the Cbf5p enzyme

    PubMed Central

    Godin, Katherine S.; Walbott, Hélène; Leulliot, Nicolas; van Tilbeurgh, Herman; Varani, Gabriele

    2009-01-01

    Box H/ACA small nucleolar ribonucleoproteins (snoRNPs) are responsible for the formation of pseudouridine in a variety of RNAs, and are essential for ribosome biogenesis, modification of spliceosomal RNAs and telomerase stability. The mature snoRNP has been reconstituted in vitro and is composed of a single RNA and four proteins. However, snoRNP biogenesis in vivo requires multiple factors to coordinate a complex and poorly understood assembly and maturation process. Among the factors required for snoRNP biogenesis in yeast is Shq1p, an essential protein necessary for the snoRNA stable expression. We have found that Shq1p consists of two independent domains that contain Casein Kinase 1 phosphorylation sites. We also demonstrate that Shq1p binds the pseudourydilating enzyme Cbf5p through the C-terminal domain in synergy with the N-terminal domain. The NMR solution structure of the N-terminal domain has striking homology to the ‘Chord and Sgt1’ (CS) domain of known Hsp90 co-chaperones, yet Shq1p does not interact with the yeast Hsp90 homologue in vitro. Surprisingly, Shq1p has stand-alone chaperone activity in vitro. This activity is harbored by the C-terminal domain but it is increased by the presence of the N-terminal domain. These results provide the first evidence of a specific biochemical activity for Shq1p and a direct link to the H/ACA snoRNP. PMID:19426738

  10. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein*

    PubMed Central

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S.; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M.; Brandes, Ralf P.

    2016-01-01

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. PMID:26861875

  11. The Collagen Binding Protein Cnm Contributes to Oral Colonization and Cariogenicity of Streptococcus mutans OMZ175

    PubMed Central

    Miller, James H.; Avilés-Reyes, Alejandro; Scott-Anne, Kathy; Gregoire, Stacy; Watson, Gene E.; Sampson, Edith; Progulske-Fox, Ann; Koo, Hyun; Bowen, William H.; Lemos, José A.

    2015-01-01

    Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm+ native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm+ isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity. PMID:25733523

  12. Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications

    PubMed Central

    2012-01-01

    Background Collagen has proved valuable as biomedical materials for a range of clinical applications, particularly in wound healing. It is normally produced from animal sources, such as from bovines, but concerns have emerged over transmission of diseases. Recombinant collagens would be preferable, but are difficult to produce. Recently, studies have shown that ‘collagens’ from bacteria, including Streptococcus pyogenes, can be produced in the laboratory as recombinant products, and that these are biocompatible. In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins. Results Production trials in shake flask cultures gave low yields of recombinant product, < 1 g/L. Increased yields, of around 1 g/L, were obtained when the shake flask process was transferred to a stirred tank bioreactor, and the yield was further enhanced to around 10 g/L by implementation of a high cell density fed-batch process and the use of suitably formulated fully defined media. Similar yields were obtained with 2 different constructs, one containing an introduced heparin binding domain. The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time. Conclusions These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications. PMID:23126526

  13. 150-kD von Willebrand factor binding protein extracted from human vascular subendothelium is type VI collagen.

    PubMed Central

    Rand, J H; Patel, N D; Schwartz, E; Zhou, S L; Potter, B J

    1991-01-01

    We have previously shown that von Willebrand factor (vWF), a glycoprotein which plays a critical role in the adhesion of platelets to injured blood vessels, is present within vascular subendothelium. We investigated the identity of the subendothelial binding site(s) for vWF by examining vWF binding to subendothelial constituents and solubilized a 150-kD protein with SDS-urea that bound vWF. This protein had an amino-acid composition similar to that of the type VI collagen alpha-1/alpha-2 chains, was recognized by specific polyclonal antibodies against type VI collagen, and had a similar acidic isoelectric point. Furthermore, we found that purified type VI collagen also bound vWF. Thus, we have identified the extracted 150-kD protein as type VI collagen. This protein may play a significant role in the binding of vWF to vascular subendothelium in vivo. Images PMID:2056120

  14. Simultaneous Platinum and Copper Ion Attachment to a Human Copper Chaperone Protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Cvitkovic, John; Yu, Corey; Dmitriev, Oleg; Kaminski, George; Bernholc, Jerry

    2015-03-01

    Cisplatin is a potent anti-cancer drug based on a platinum ion. However, its effectiveness is decreased by cellular resistance, which involves cisplatin attaching to copper transport proteins. One of such proteins is Atox1, where cisplatin attaches to the copper binding site. Surprisingly, it was shown that both cisplatin and copper can attach to Atox1 at the same time. To study this double metal ion attachment, we use the KS/FD DFT method, which combines Kohn-Sham DFT with frozen-density DFT to achieve efficient quantum-mechanical description of explicit solvent. Calculations have so far investigated copper ion attachment to CXXC motifs present in Atox1. The addition of the platinum ion and the competition between the two metals is currently being studied. These calculations start from a molecular mechanics (MM) structural model, in which glutathione groups provide additional ligands to the Pt ion. Our goals are to identify possible Cu-Pt structures and to determine whether copper/platinum attachment is competitive, independent, or cooperative. Results will be compared to the 1H, N1 5 -HSQC NMR experiments, in which binding of copper and cisplatin to Atox1 produces distinct secondary chemical shift signatures, allowing for kinetic studies of simultaneous metal binding.

  15. 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

  16. Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

    PubMed Central

    Eyre, David R.; Ann Weis, Mary

    2013-01-01

    Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

  17. 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

  18. 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

  19. Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD)

    PubMed Central

    Kamma-Lorger, Christina S.; Pinali, Christian; Martínez, Juan Carlos; Harris, Jon; Young, Robert D.; Bredrup, Cecilie; Crosas, Eva; Malfois, Marc; Rødahl, Eyvind

    2016-01-01

    The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal. PMID:26828927

  20. Bioengineered collagens

    PubMed Central

    Ramshaw, John AM; Werkmeister, Jerome A; Dumsday, Geoff J

    2014-01-01

    Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications. PMID:24717980

  1. Remodeling of the heart (membrane proteins and collagen) in hypertensive cardiopathy.

    PubMed

    Sainte Beuve, C; Leclercq, C; Rannou, F; Oliviero, P; Mansier, P; Chevalier, B; Swynghedauw, B; Charlemagne, D

    1992-06-01

    The basis for impaired left ventricular function of hearts in moderate to severe stages of hypertrophy and congestive heart failure remains uncertain. At the cellular level, the mechanisms governing the movements of calcium in the myocardium are actually depressed and might at least in part account for the slowing of the maximum shortening velocity and the impaired relaxation. These alterations of membrane proteins seem particularly important in species where the slowing of Vmax cannot be a consequence of the myosin heavy chain shift. They lead to an unstable equilibrium of calcium homeostasis and to calcium overload in heart failure. On the other hand, the enhanced density and remodeling of collagen in the hypertrophied heart, which would depend on elevation in circulating aldosterone, impair myocardial stiffness with diastolic dysfunction and lead to altered pumping capacity of the heart. Disturbances of calcium metabolism and matrix collagen remodeling enhance early afterdepolarizations and arrhythmias. PMID:1385839

  2. Differential Loss of Prolyl Isomerase or Chaperone Activity of Ran-binding Protein 2 (Ranbp2) Unveils Distinct Physiological Roles of Its Cyclophilin Domain in Proteostasis*

    PubMed Central

    Cho, Kyoung-in; Patil, Hemangi; Senda, Eugene; Wang, Jessica; Yi, Haiqing; Qiu, Sunny; Yoon, Dosuk; Yu, Minzhong; Orry, Andrew; Peachey, Neal S.; Ferreira, Paulo A.

    2014-01-01

    The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2WT-HA) or without PPIase activities (Tg-Ranbp2R2944A-HA). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2R2944A-HA::Ranbp2−/−. Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2R2944A-HA::Ranbp2−/−. This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2CLDm-HA::Ranbp2−/−, harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil

  3. ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-inducible factor (HIF)-1 transcriptional activity and heat shock protein (HSP) 60 chaperon activity.

    PubMed

    Li, Guangzhe; Azuma, Soyoko; Sato, Shinichi; Minegishi, Hidemitsu; Nakamura, Hiroyuki

    2015-07-01

    ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds synthesized, compound 1d showed the most significant inhibition of hypoxia-induced HIF-1 transcriptional activity with the IC50 of 0.53μM. Furthermore, compound 1h was found to possess the most significant inhibition of heat shock protein (HSP) 60 chaperon activity among the reported inhibitors: the IC50 toward the porcine heart malate dehydrogenase (MDH) refolding assay was 0.35μM. PMID:25981686

  4. Cervical Softening During Pregnancy: Regulated Changes in Collagen Cross-Linking and Composition of Matricellular Proteins in the Mouse1

    PubMed Central

    Akins, Meredith L.; Luby-Phelps, Katherine; Bank, Ruud A.; Mahendroo, Mala

    2011-01-01

    A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C. PMID:21248285

  5. Fundamental Investigations of the Extracellular Proteins Fibrin and Collagen in Microchannel Devices

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Koester, Sarah; Pfohl, Thomas

    2007-03-01

    Microfluidic structures are particularly amenable to controlled investigations of protein bundle and network formation. Hydrodynamic focusing is utilized to create a diffusion-controlled gradient of reactants, enabling non-equilibrium investigations. We present studies of the blood clotting protein fibrin, a three-dimensional network formed from the enzymatic cleavage of fibrinogen monomers by the protein thrombin. Fibrin is a vital component of blood clots, and has been implicated in a variety of diseases. Real-time fluorescence microscopy and x-ray micro-diffraction are used to quantify supramolecular assembly and provide snapshots of the evolution of fibrin network formation. We also show that collagen, a ubiquitous extracellular protein, can be bundled in situ through the use of a pH gradient. An outlook toward artificial blood vessels arises from the insight that both fibrin and collagen can easily be used to coat microchannel structures. The resulting mesh forms an ideal environment for red blood cells and other cell types.

  6. Asymmetry adjacent to the collagen-like domain in rat liver mannose-binding protein.

    PubMed Central

    Wallis, R; Drickamer, K

    1997-01-01

    Rat liver mannose-binding protein (MBP-C) is the smallest known member of the collectin family of animal lectins, many of which are involved in defence against microbial pathogens. It consists of an N-terminal collagen-like domain linked to C-terminal carbohydrate-recognition domains. MBP-C, overproduced in Chinese-hamster ovary cells, is post-translationally modified and processed in a manner similar to the native lectin. Analytical ultracentrifugation experiments indicate that MBP-C is trimeric, with a weight-averaged molecular mass of approx. 77 kDa. The rate of sedimentation of MBP-C and its mobility on gel filtration suggest a highly elongated molecule. Anomalous behaviour on gel filtration due to this extended conformation may explain previous suggestions that MBP-C forms a higher oligomer. The polypeptide chains of the MBP-C trimer are linked by disulphide bonds between two cysteine residues at the N-terminal junction of the collagen-like domain. Analysis of an N-terminal tryptic fragment reveals that the disulphide bonding in MBP-C is heterogeneous and asymmetrical. These results indicate that assembly of MBP-C oligomers probably proceeds in a C- to N-terminal direction: trimerization at the C-terminus is followed by assembly of the collagenous domain and finally formation of N-terminal disulphide bonds. The relatively simple organization of MBP-C provides a template for understanding larger, more complex collectins. PMID:9230118

  7. 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

  8. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.

    PubMed

    Sha, Chong; Yu, Xiao-Wei; Lin, Nai-Xin; Zhang, Meng; Xu, Yan

    2013-12-10

    Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris. PMID:24315648

  9. The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3

    PubMed Central

    Drané, Pascal; Ouararhni, Khalid; Depaux, Arnaud; Shuaib, Muhammad; Hamiche, Ali

    2010-01-01

    The histone variant H3.3 marks active chromatin by replacing the conventional histone H3.1. In this study, we investigate the detailed mechanism of H3.3 replication-independent deposition. We found that the death domain-associated protein DAXX and the chromatin remodeling factor ATRX (α-thalassemia/mental retardation syndrome protein) are specifically associated with the H3.3 deposition machinery. Bacterially expressed DAXX has a marked binding preference for H3.3 and assists the deposition of (H3.3–H4)2 tetramers on naked DNA, thus showing that DAXX is a H3.3 histone chaperone. In DAXX-depleted cells, a fraction of H3.3 was found associated with the replication-dependent machinery of deposition, suggesting that cells adapt to the depletion. The reintroduced DAXX in these cells colocalizes with H3.3 into the promyelocytic leukemia protein (PML) bodies. Moreover, DAXX associates with pericentric DNA repeats, and modulates the transcription from these repeats through assembly of H3.3 nucleosomes. These findings establish a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure. Taken together, our data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3. PMID:20504901

  10. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

    PubMed

    Cahoon, Laty A; Freitag, Nancy E; Prehna, Gerd

    2016-07-01

    Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates. PMID:27007641

  11. 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.

  12. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

    PubMed Central

    Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

  13. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair.

    PubMed

    Caves, Jeffrey M; Cui, Wanxing; Wen, Jing; Kumar, Vivek A; Haller, Carolyn A; Chaikof, Elliot L

    2011-08-01

    Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23-314%), six-fold in Young's modulus (5.3-33.1 MPa), and more than two-fold in tensile strength (1.85-4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity. PMID:21550111

  14. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair

    PubMed Central

    Caves, Jeffrey M.; Cui, Wanxing; Wen, Jing; Kumar, Vivek A.; Haller, Carolyn A.; Chaikof, Elliot L.

    2011-01-01

    Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23 – 314%), six-fold in Young’s modulus (5.3 to 33.1 MPa), and more than two-fold in tensile strength (1.85 to 4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity. PMID:21550111

  15. Experimentally guided structural modeling and dynamics analysis of Hsp90-p53 interactions: allosteric regulation of the Hsp90 chaperone by a client protein.

    PubMed

    Blacklock, Kristin; Verkhivker, Gennady M

    2013-11-25

    A fundamental role of the Hsp90 chaperone system in mediating maturation of protein clients is essential for the integrity of signaling pathways involved in cell cycle control and organism development. Molecular characterization of Hsp90 interactions with client proteins is fundamental to understanding the activity of many tumor-inducing signaling proteins and presents an active area of structural and biochemical studies. In this work, we have probed mechanistic aspects of allosteric regulation of Hsp90 by client proteins via a detailed computational study of Hsp90 interactions with the tumor suppressor protein p53. Experimentally guided protein docking and molecular dynamics structural refinement have reconstructed the recognition-competent states of the Hsp90-p53 complexes that are consistent with the NMR studies. Protein structure network analysis has identified critical interacting networks and specific residues responsible for structural integrity and stability of the Hsp90-p53 complexes. Coarse-grained modeling was used to characterize the global dynamics of the regulatory complexes and map p53-induced changes in the conformational equilibrium of Hsp90. The variations in the functional dynamics profiles of the Hsp90-p53 complexes are consistent with the NMR studies and could explain differences in the functional role of the alternative binding sites. Despite the overall similarity of the collective movements and the same global interaction footprint, p53 binding at the C-terminal interaction site of Hsp90 may have a more significant impact on the chaperone dynamics, which is consistent with the stronger allosteric effect of these interactions revealed by the experimental studies. The results suggest that p53-induced modulation of the global dynamics and structurally stable interaction networks can target the regulatory hinge regions and facilitate stabilization of the closed Hsp90 dimer that underlies the fundamental stimulatory effect of the p53 client. PMID

  16. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  17. 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

  18. Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

    PubMed Central

    Yao, Y; Zhou, Y; Wang, C

    1997-01-01

    The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI. PMID:9034346

  19. E-spun composite fibers of collagen and dragline silk protein: fiber mechanics, biocompatibility, and application in stem cell differentiation.

    PubMed

    Zhu, Bofan; Li, Wen; Lewis, Randolph V; Segre, Carlo U; Wang, Rong

    2015-01-12

    Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen-silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices. PMID:25405355

  20. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  1. Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2▿

    PubMed Central

    Miki, Tsuyoshi; Shibagaki, Yoshio; Danbara, Hirofumi; Okada, Nobuhiko

    2009-01-01

    The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery. PMID:19767440

  2. Streptococcus gordonii collagen-binding domain protein CbdA may enhance bacterial survival in instrumented root canals ex vivo

    PubMed Central

    Moses, Peter J.; Power, Daniel A.; Jesionowski, Amy M.; Jenkinson, Howard F.; Pantera, Eugene A.; Vickerman, M. Margaret

    2012-01-01

    Introduction The surface-associated collagen-binding protein Ace of Enterococcus faecalis has been implicated as a virulence factor that contributes to bacterial persistence in endodontic infections. The purpose of this study was to determine if proteins with amino acid sequence similarity to Ace found in more abundant oral streptococci could play a similar role in potentially enhancing endodontic infections. Methods A Streptococcus gordonii gene similar to ace was identified by genome sequence searches in silico. An isogenic derivative of strain DL1 with a disruption in the identified gene was constructed by allelic replacement. Parent and mutant strains were characterized for their ability to bind immobilized collagen type-1 in a microtiter plate binding assay. Survival of the strains in a human tooth ex vivo instrumented root canal model was compared by inoculating canals with parental or mutant bacteria and determining the CFUs recovered at various time points over a 12-day period. Results The S. gordonii gene, encoding a protein with a conserved collagen-binding domain similar to that of Ace, was designated cbdA. The cbdA-deficient cells were less able to bind collagen type-1 than parental cells (P <0.0001). Genetic complementation of the cbdA-deficient strain restored the collagen-binding phenotype. By day 12 significantly fewer (P =0.03) cbdA-deficient than parental CFUs were recovered from instrumented canals. Conclusion A gene encoding a putative collagen-binding protein was identified in S. gordonii. Fewer S. gordonii cbdA-deficient cells survived ex vivo compared with parental cells, suggesting that collagen-binding proteins may contribute to persistence of oral streptococci in instrumented root canals. PMID:23228255

  3. The Collagen Family

    PubMed Central

    Ricard-Blum, Sylvie

    2011-01-01

    Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions. PMID:21421911

  4. [Determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-mass spectrometry].

    PubMed

    Xia, Jingen; Chen, Bo; Yao, Shouzhuo

    2008-09-01

    A simple, rapid and accurate method for the determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was developed. After hydrolysis of collagenous proteins with hydrochloric acid, the hydroxyproline was separated on a Spherigel C8 column using acetonitrile-0.05% trifluoroacetic acid aqueous solution (5:95, v/v) at a flow rate of 1.0 mL/min. Theanine was used as the internal standard for the quantification. Hydroxyproline was identified and quantified by electrospray ionization mass spectrometry (ESI-MS), which was operated in positive ion mode while the m/z 132 and m/z 175 ions were recorded in the selective ionization monitoring (SIM) mode. The peak area ratio of hydroxyproline to theanine versus the hydroxyproline concentration was linear over a concentration range of 11.7-117 mg/L for hydroxyproline. The correlation coefficient was 0.999 3. The reproducibility and average recovery of the method were 1.87% and 98.85% respectively. The method has the advantages of easy operation, without derivatization, less analysis time, good precision and accuracy. PMID:19160759

  5. 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

  6. 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

  7. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  8. 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

  9. Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

    PubMed Central

    Godet, Julien; Kenfack, Cyril; Przybilla, Frédéric; Richert, Ludovic; Duportail, Guy; Mély, Yves

    2013-01-01

    The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway. PMID:23511968

  10. Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones1[C][W

    PubMed Central

    Merino, Irene; Contreras, Angela; Jing, Zhong-Ping; Gallardo, Fernando; Cánovas, Francisco M.; Gómez, Luis

    2014-01-01

    Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula × Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones. PMID:24306533

  11. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    SciTech Connect

    Walker, G.; Bourguignon, L.Y. )

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  12. E-Spun Composite Fibers of Collagen and Dragline Silk Protein: Fiber Mechanics, Biocompatibility, and Application in Stem Cell Differentiation

    PubMed Central

    2015-01-01

    Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen–silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices. PMID:25405355

  13. Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin.

    PubMed

    Gistelinck, C; Gioia, R; Gagliardi, A; Tonelli, F; Marchese, L; Bianchi, L; Landi, C; Bini, L; Huysseune, A; Witten, P E; Staes, A; Gevaert, K; De Rocker, N; Menten, B; Malfait, F; Leikin, S; Carra, S; Tenni, R; Rossi, A; De Paepe, A; Coucke, P; Willaert, A; Forlino, A

    2016-01-01

    Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635

  14. Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin

    PubMed Central

    Gistelinck, C.; Gioia, R.; Gagliardi, A.; Tonelli, F.; Marchese, L.; Bianchi, L.; Landi, C.; Bini, L.; Huysseune, A.; Witten, P. E.; Staes, A.; Gevaert, K.; De Rocker, N.; Menten, B.; Malfait, F.; Leikin, S.; Carra, S.; Tenni, R.; Rossi, A.; De Paepe, A.; Coucke, P.; Willaert, A.; Forlino, A.

    2016-01-01

    Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635

  15. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    PubMed

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. PMID:17661353

  16. Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella.

    PubMed

    Fardini, Yann; Trotereau, Jérôme; Bottreau, Elisabeth; Souchard, Charlène; Velge, Philippe; Virlogeux-Payant, Isabelle

    2009-05-01

    In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the DeltabamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in DeltabamC and DeltadegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As DeltasurA and DeltabamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD. PMID:19372159

  17. 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

  18. 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

  19. The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices.

    PubMed

    Chen, J-S; Noah, E M; Pallua, N; Steffens, G C M

    2002-11-01

    The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins - horse radish peroxidase (HRP) was used as a model protein - were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP-PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices. PMID:15348172

  20. Chaperone Hsp27, a Novel Subunit of AUF1 Protein Complexes, Functions in AU-Rich Element-Mediated mRNA Decay▿ †

    PubMed Central

    Sinsimer, Kristina S.; Gratacós, Frances M.; Knapinska, Anna M.; Lu, Jiebo; Krause, Christopher D.; Wierzbowski, Alexandria V.; Maher, Lauren R.; Scrudato, Shannon; Rivera, Yonaira M.; Gupta, Swati; Turrin, Danielle K.; De La Cruz, Mary Pauline; Pestka, Sidney; Brewer, Gary

    2008-01-01

    Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3′ untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility. PMID:18573886

  1. The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone.

    PubMed

    Konieczny, I; Helinski, D R

    1997-12-23

    Initiation and control of replication of the broad-host-range plasmid RK2 requires two plasmid-encoded elements, the replication origin (oriV) and the initiation protein TrfA. Purified TrfA is largely in the form of a dimer; however, only the monomeric form of the protein can bind specifically to the direct repeats (iterons) at the RK2 origin. The largely dimeric form of wild-type TrfA is inactive in the initiation of replication of RK2 in an in vitro replication system reconstituted from purified components. However, preincubation of the TrfA protein with the ClpX molecular chaperone isolated from Escherichia coli activates the initiator protein for replication in the purified system. We further observed that ClpX, in an ATP-dependent reaction, greatly increases the proportion of TrfA monomers and, therefore, the ability of this protein to bind to iterons localized within RK2 origin. Finally, a copy-up mutant of the TrfA protein which is largely in the monomer form is active in the reconstituted in vitro replication system, and its activity is not affected by ClpX. PMID:9405620

  2. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  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. The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke

    PubMed Central

    Nakano, Kazuhiko; Hokamura, Kazuya; Taniguchi, Naho; Wada, Koichiro; Kudo, Chiho; Nomura, Ryota; Kojima, Ayuchi; Naka, Shuhei; Muranaka, Yoshinori; Thura, Min; Nakajima, Atsushi; Masuda, Katsuhiko; Nakagawa, Ichiro; Speziale, Pietro; Shimada, Nobumitsu; Amano, Atsuo; Kamisaki, Yoshinori; Tanaka, Tokutaro; Umemura, Kazuo; Ooshima, Takashi

    2011-01-01

    Although several risk factors for stroke have been identified, one-third remain unexplained. Here we show that infection with Streptococcus mutans expressing collagen-binding protein (CBP) is a potential risk factor for haemorrhagic stroke. Infection with serotype k S. mutans, but not a standard strain, aggravates cerebral haemorrhage in mice. Serotype k S. mutans accumulates in the damaged, but not the contralateral hemisphere, indicating an interaction of bacteria with injured blood vessels. The most important factor for high-virulence is expression of CBP, which is a common property of most serotype k strains. The detection frequency of CBP-expressing S. mutans in haemorrhagic stroke patients is significantly higher than in control subjects. Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated. Administration of recombinant CBP causes aggravation of haemorrhage. Our data suggest that CBP of S. mutans is directly involved in haemorrhagic stroke. PMID:21952219

  5. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    PubMed

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes. PMID:25118328

  6. 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

  7. 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

  8. OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood–Brain Barrier: Implications for Anti-Cancer Therapies

    PubMed Central

    BOOTH, LAURENCE; ROBERTS, JANE L.; TAVALLAI, MEHRAD; NOURBAKHSH, AIDA; CHUCKALOVCAK, JOHN; CARTER, JORI; POKLEPOVIC, ANDREW; DENT, PAUL

    2016-01-01

    We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. PMID:25736380

  9. Chaperone Role for Proteins p618 and p892 in the Extracellular Tail Development of Acidianus Two-Tailed Virus ▿ †

    PubMed Central

    Scheele, Urte; Erdmann, Susanne; Ungewickell, Ernst J.; Felisberto-Rodrigues, Catarina; Ortiz-Lombardía, Miguel; Garrett, Roger A.

    2011-01-01

    The crenarchaeal Acidianus two-tailed virus (ATV) undergoes a remarkable morphological development, extracellularly and independently of host cells, by growing long tails at each end of a spindle-shaped virus particle. Initial work suggested that an intermediate filament-like protein, p800, is involved in this process. We propose that an additional chaperone system is required, consisting of a MoxR-type AAA ATPase (p618) and a von Willebrand domain A (VWA)-containing cochaperone, p892. Both proteins are absent from the other known bicaudavirus, STSV1, which develops a single tail intracellularly. p618 exhibits ATPase activity and forms a hexameric ring complex that closely resembles the oligomeric complex of the MoxR-like protein RavA (YieN). ATV proteins p387, p653, p800, and p892 interact with p618, and with the exception of p800, all bind to DNA. A model is proposed to rationalize the interactions observed between the different protein and DNA components and to explain their possible structural and functional roles in extracellular tail development. PMID:21367903

  10. Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths.

    PubMed

    Tohse, Hidekazu; Takagi, Yasuaki; Nagasawa, Hiromichi

    2008-05-01

    In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral-associated proteins. To identify such proteins, we have searched for proteins that may form high-molecular-weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule-64 (OMM-64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu-rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [Söllner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282-286]. (45)Ca overlay analysis revealed that the Glu-rich region has calcium-binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM-64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM-64 is expressed specifically in otolith matrix-producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear-specific short-chain collagen otolin-1, and the resulting complex forms ring-like structures in the otolith matrix. Overall, OMM-64, by forming a calcium-binding aggregate that binds to otolin-1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization. PMID:18410381

  11. Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

    PubMed

    Homan, Erica P; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M; Dawson, Brian; Bertin, Terry K; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H L

    2014-01-01

    Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

  12. Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

    PubMed Central

    Homan, Erica P.; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M.; Dawson, Brian; Bertin, Terry K.; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H. L.

    2014-01-01

    Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

  13. NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins.

    PubMed

    Bizarro, Jonathan; Dodré, Maxime; Huttin, Alexandra; Charpentier, Bruno; Schlotter, Florence; Branlant, Christiane; Verheggen, Céline; Massenet, Séverine; Bertrand, Edouard

    2015-10-15

    The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins. PMID:26275778

  14. NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins

    PubMed Central

    Bizarro, Jonathan; Dodré, Maxime; Huttin, Alexandra; Charpentier, Bruno; Schlotter, Florence; Branlant, Christiane; Verheggen, Céline; Massenet, Séverine; Bertrand, Edouard

    2015-01-01

    The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins. PMID:26275778

  15. 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

  16. Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

    PubMed

    Schröder, H C; Krasko, A; Batel, R; Skorokhod, A; Pahler, S; Kruse, M; Müller, I M; Müller, W E

    2000-10-01

    The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system. PMID:11023986

  17. Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions

    PubMed Central

    Kim, Young Kyung; Gu, Li-sha; Bryan, Thomas E.; Kim, Jong Ryul; Chen, Liang; Liu, Yan; Yoon, James C.; Breschi, Lorenzo; Pashley, David H.; Tay, Franklin R.

    2010-01-01

    The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the nonclassical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 hours after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes. PMID:20621767

  18. Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions.

    PubMed

    Kim, Young Kyung; Gu, Li-sha; Bryan, Thomas E; Kim, Jong R; Chen, Liang; Liu, Yan; Yoon, James C; Breschi, Lorenzo; Pashley, David H; Tay, Franklin R

    2010-09-01

    The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the non-classical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 h after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes. PMID:20621767

  19. 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

  20. 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

  1. Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone

    PubMed Central

    Hussein, Rasha M.; Hashem, Reem M.; Rashed, Laila A.

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ) peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP) that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases) both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of human embryonic kidney 293 (HEK293) cells with Aβ-green fluorescent protein (GFP) fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly glutamine (Poly Q) peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells. PMID:26283911

  2. Action of Multiple Endoplasmic Reticulum Chaperon-like Proteins Is Required for Proper Folding and Polarized Localization of Kre6 Protein Essential in Yeast Cell Wall β-1,6-Glucan Synthesis*

    PubMed Central

    Kurita, Tomokazu; Noda, Yoichi; Yoda, Koji

    2012-01-01

    Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis. PMID:22447934

  3. SdrX, a serine-aspartate repeat protein expressed by Staphylococcus capitis with collagen VI binding activity.

    PubMed

    Liu, Yule; Ames, Brenda; Gorovits, Elena; Prater, Bradley D; Syribeys, Peter; Vernachio, John H; Patti, Joseph M

    2004-11-01

    Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis. PMID:15501749

  4. A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure

    PubMed Central

    Dong, Shaojian; Jin, Can; Li, Jiale

    2015-01-01

    In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer. PMID:26262686

  5. COLLAGEN STRUCTURE AND STABILITY

    PubMed Central

    Shoulders, Matthew D.; Raines, Ronald T.

    2010-01-01

    Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed polyproline II-type helices. Much progress has been made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen–the prototypical collagen fibril–has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology. PMID:19344236

  6. FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones.

    PubMed

    Pan, Tai-Long; Hsu, Shu-Yuan; Wang, Pei-Wen; Cheng, Ya-Ting; Chang, Yu-Chen; Saha, Sudipta; Hu, Jiwei; Ouyang, Pin

    2015-01-01

    Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome over-replication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxic-stress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. The results of this study pave the way to our further understanding of the role of this novel cytokinesis-related protein in protecting cells from environmental stress and tetraploid formation. PMID:25751302

  7. Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK*

    PubMed Central

    Albareda, Marta; Pacios, Luis F.; Manyani, Hamid; Rey, Luis; Brito, Belén; Imperial, Juan; Ruiz-Argüeso, Tomás; Palacios, Jose M.

    2014-01-01

    [NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O2) and bacteroids from legume nodules (∼10–100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen. PMID:24942742

  8. Molecular chaperone heat shock protein 70 participates in the labile phase of the development of behavioural sensitization induced by a single morphine exposure in mice.

    PubMed

    Qin, Wang-Jun; Wang, Yan-Ting; Zhang, Min; Wen, Rui-Ting; Liu, Qing; Li, Yu-Ling; Chen, Feng; Lawrence, Andrew J; Liang, Jian-Hui

    2013-04-01

    De-novo protein synthesis is required in the development of behavioural sensitization. A prior screening test from our laboratory has implicated heat shock protein 70 (Hsp70) as one of the proteins required in this behavioural plasticity. Thus, this study was designed to extend our understanding of the role of Hsp70 in the development of behavioural sensitization induced by a single morphine exposure in mice. First, by employing transcription inhibitor actinomycin D (AD) and protein synthesis inhibitor cycloheximide (CHX), we identified a protein synthesis-dependent labile phase (within 4 h after the first morphine injection) in the development of behavioural sensitization to a single morphine exposure. Second, Hsp70 protein expression in the nucleus accumbens correlated positively with locomotor responses of sensitized mice and, more importantly, the expression of Hsp70 increased within 1 h after the first morphine injection. Third, AD and CHX both prevented expression of Hsp70 and disrupted the development of the single morphine induced behavioural sensitization, which further implied Hsp70 was highly associated with behavioural sensitization. Finally, the selective Hsp70 inhibitor pifithrin-μ (PES) i.c.v. injected in mice prevented the development of behavioural sensitization and, critically, this inhibitory effect occurred only when PES was given within 1 h after the first morphine injection, which was within the labile phase of the development period. Taken together, we draw the conclusion that Hsp70 is crucially involved in the labile phase of the development of behavioural sensitization induced by a single morphine exposure, probably functioning as a molecular chaperone. PMID:22647551

  9. Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen.

    PubMed

    Hill, Ryan C; Wither, Matthew J; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C

    2015-07-01

    Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼ 120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757

  10. Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen*

    PubMed Central

    Hill, Ryan C.; Wither, Matthew J.; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C.

    2015-01-01

    Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757

  11. COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen VI congenital muscular dystrophy.

    PubMed

    Pan, Te-Cheng; Zhang, Rui-Zhu; Markova, Dessislava; Arita, Machiko; Zhang, Yejia; Bogdanovich, Sasha; Khurana, Tejvir S; Bönnemann, Carsten G; Birk, David E; Chu, Mon-Li

    2013-05-17

    Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a Col6a3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The Col6a3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy. PMID:23564457

  12. Collagen peptide-based biomaterials for protein delivery and peptide-promoted self-assembly of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Ernenwein, Dawn M.

    2011-12-01

    Bottom-up self-assembly of peptides has driven the research progress for the following two projects: protein delivery vehicles of collagen microflorettes and the assembly of gold nanoparticles with coiled-coil peptides. Collagen is the most abundant protein in the mammals yet due to immunogenic responses, batch-to-batch variability and lack of sequence modifications, synthetic collagen has been designed to self-assemble into native collagen-like structures. In particular with this research, metal binding ligands were incorporated on the termini of collagen-like peptides to generate micron-sized particles, microflorettes. The over-arching goal of the first research project is to engineer MRI-active microflorettes, loaded with His-tagged growth factors with differential release rates while bound to stem cells that can be implemented toward regenerative cell-based therapies. His-tagged proteins, such as green fluorescent protein, have successfully been incorporated on the surface and throughout the microflorettes. Protein release was monitored under physiological conditions and was related to particle degradation. In human plasma full release was obtained within six days. Stability of the microflorettes under physiological conditions was also examined for the development of a therapeutically relevant delivery agent. Additionally, MRI active microflorettes have been generated through the incorporation of a gadolinium binding ligand, DOTA within the collagen-based peptide sequence. To probe peptide-promoted self-assemblies of gold nanoparticles (GNPs) by non-covalent, charge complementary interactions, a highly anionic coiled-coil peptide was designed and synthesized. Upon formation of peptide-GNP interactions, the hydrophobic domain of the coiled-coil were shown to promote the self-assembly of peptide-GNPs clustering. Hydrophobic forces were found to play an important role in the assembly process, as a peptide with an equally overall negative charge, but lacking an

  13. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo. PMID:24798087

  14. Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-05-03

    The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

  15. NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease.

    PubMed

    Karradt, Anne; Sobanski, Johanna; Mattow, Jens; Lockau, Wolfgang; Baier, Kerstin

    2008-11-21

    When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the alpha-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC.ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process. PMID:18818204

  16. A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage

    PubMed Central

    Pham, Bang Phuong; Kwak, Jae Myeong; Xuan, Yuan Hu; Cheong, Gang-Won

    2015-01-01

    Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys46) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys205 and Cys211) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx. PMID:25933432

  17. The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation, Autoaggregation, and Pathogen-Phagocyte Interactions

    PubMed Central

    Abdel-Nour, Mena; Duncan, Carla; Prashar, Akriti; Rao, Chitong; Ginevra, Christophe; Jarraud, Sophie; Low, Donald E.; Ensminger, Alexander W.; Terebiznik, Mauricio R.

    2014-01-01

    Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae. PMID:24334670

  18. 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.

  19. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

    PubMed

    Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2016-01-01

    Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel. PMID:25005915

  20. Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

    PubMed Central

    Pasculescu, Adrian; Dai, Anna Yue; Williton, Kelly; Taylor, Lorne; Savitski, Mikhail M.; Bantscheff, Marcus; Woodgett, James R.; Pawson, Tony; Colwill, Karen

    2016-01-01

    The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. PMID:26755559

  1. Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

    PubMed Central

    Xue, Niannan; Li, Xia; Bertulli, Cristina; Li, Zhaoying; Patharagulpong, Atipat; Sadok, Amine; Huang, Yan Yan Shery

    2014-01-01

    Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among

  2. Atomic force microscopy reveals a dual collagen-binding activity for the staphylococcal surface protein SdrF.

    PubMed

    Herman-Bausier, Philippe; Dufrêne, Yves F

    2016-02-01

    Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall-anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine-aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single-cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen-coated substrates through both weak and strong bonds. Single-molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand-binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well-characterized 'dock, lock and latch' mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins. PMID:26481199

  3. A mouse 3T6 fibroblast cell culture model for the study of normal and protein-engineered collagen synthesis and deposition into the extracellular matrix.

    PubMed

    Lamandé, S R; Bateman, J F

    1993-07-01

    Mouse 3T6 fibroblasts deposited an organized collagenous extracellular matrix during long-term culture in the presence of ascorbic acid. The matrix produced by the cells had a similar distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III and V collagens. By day 8 of culture more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the alpha 1 (I)CB6 and alpha 2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesised collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable matrix present, 29% of the collagen synthesised was deposited, while by day 15, 88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix. PMID:8412990

  4. Effects of cis-4-hydroxy-L-proline, an inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

    SciTech Connect

    Eldridge, C.F.; Bunge, R.P.; Bunge, M.B. )

    1988-02-01

    The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. The authors confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen. Cultures grown in the presence or absence of CHP were metabolically labeled with ({sup 3}H)leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. They conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. They discuss the evidence for and against specificity of CHP action in other systems.

  5. Softenin, a Novel Protein That Softens the Connective Tissue of Sea Cucumbers through Inhibiting Interaction between Collagen Fibrils

    PubMed Central

    Takehana, Yasuhiro; Yamada, Akira; Tamori, Masaki; Motokawa, Tatsuo

    2014-01-01

    The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between

  6. Aryl Hydrocarbon Receptor-interacting Protein-like 1 Is an Obligate Chaperone of Phosphodiesterase 6 and Is Assisted by the γ-Subunit of Its Client.

    PubMed

    Gopalakrishna, Kota N; Boyd, Kimberly; Yadav, Ravi P; Artemyev, Nikolai O

    2016-07-29

    Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory Pγ-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and Pγ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants. PMID:27268253

  7. Serological detection of ‘Candidatus Liberibacter asiaticus’ in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens

    PubMed Central

    Ding, Fang; Duan, Yongping; Yuan, Qing; Shao, Jonathan; Hartung, John S.

    2016-01-01

    We describe experiments with antibodies against ‘Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens. PMID:27381064

  8. Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens.

    PubMed

    Ding, Fang; Duan, Yongping; Yuan, Qing; Shao, Jonathan; Hartung, John S

    2016-01-01

    We describe experiments with antibodies against 'Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens. PMID:27381064

  9. The amino terminus of the F1-ATPase beta-subunit precursor functions as an intramolecular chaperone to facilitate mitochondrial protein import.

    PubMed Central

    Hájek, P; Koh, J Y; Jones, L; Bedwell, D M

    1997-01-01

    Mitochondrial import signals have been shown to function in many steps of mitochondrial protein import. Previous studies have shown that the F1-ATPase beta-subunit precursor (pre-F1beta) of the yeast Saccharomyces cerevisiae contains an extended, functionally redundant mitochondrial import signal at its amino terminus. However, the full significance of this functionally redundant targeting sequence has not been determined. We now report that the extended pre-F1beta signal acts to maintain the precursor in an import-competent conformation prior to import, in addition to its previously characterized roles in mitochondrial targeting and translocation. We found that this extended signal is required for the efficient posttranslational mitochondrial import of pre-F1beta both in vivo and in vitro. To determine whether the pre-F1beta signal directly influences precursor conformation, fusion proteins that contain wild-type and mutant forms of the pre-F1beta import signal attached to the model passenger protein dihydrofolate reductase (DHFR) were constructed. Deletions that reduced the import signal to a minimal functional unit decreased both the half-time of precursor folding and the efficiency of mitochondrial import. To confirm that the reduced mitochondrial import associated with this truncated signal was due to a defect in its ability to maintain DHFR in a loosely folded conformation, we introduced structurally destabilizing missense mutations into the DHFR passenger to block precursor folding independently of the import signal. We found that the truncated signal imported this destabilized form of DHFR as efficiently as the intact targeting signal, indicating that the primary defect associated with the minimal signal is an inability to maintain the precursor in a loosely folded conformation. Our results suggest that the loss of this intramolecular chaperone function leads to defects in the early stages of the import process. PMID:9372949

  10. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer’s disease

    PubMed Central

    Chang, Kuo-Hsuan; Chen, I-Cheng; Lin, Hsuan-Yuan; Chen, Hsuan-Chiang; Lin, Chih-Hsin; Lin, Te-Hsien; Weng, Yu-Ting; Chao, Chih-Ying; Wu, Yih-Ru; Lin, Jung-Yaw; Lee-Chen, Guey-Jen; Chen, Chiung-Mei

    2016-01-01

    Background Alzheimer’s disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. Materials and methods Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. Results Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. Conclusion This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification. PMID:27013866

  11. Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

    PubMed

    Heidebrecht, F; Schulz, I; Keller, M; Behrens, S-E; Bader, A

    2009-10-01

    The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture. PMID:19539596

  12. The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility.

    PubMed

    Mitzelfelt, Katie A; Limphong, Pattraranee; Choi, Melinda J; Kondrat, Frances D L; Lai, Shuping; Kolander, Kurt D; Kwok, Wai-Meng; Dai, Qiang; Grzybowski, Michael N; Zhang, Huali; Taylor, Graydon M; Lui, Qiang; Thao, Mai T; Hudson, Judith A; Barresi, Rita; Bushby, Kate; Jungbluth, Heinz; Wraige, Elizabeth; Geurts, Aron M; Benesch, Justin L P; Riedel, Michael; Christians, Elisabeth S; Minella, Alex C; Benjamin, Ivor J

    2016-07-15

    Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an early-onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals. PMID:27226619

  13. IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone*

    PubMed Central

    Cracan, Valentin; Padovani, Dominique; Banerjee, Ruma

    2010-01-01

    Coenzyme B12 is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases. We designate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5′-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation. PMID:19864421

  14. The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

    PubMed Central

    Mohanan, Vishnu; Grimes, Catherine Leimkuhler

    2014-01-01

    Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. PMID:24790089

  15. A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial

    PubMed Central

    Peng, Yong Y.; Yoshizumi, Ayumi; Danon, Stephen J.; Glattauer, Veronica; Prokopenko, Olga; Mirochnitchenko, Oleg; Yu, Zhuoxin; Inouye, Masayori; Werkmeister, Jerome A.; Brodsky, Barbara; Ramshaw, John A.M.

    2011-01-01

    A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not illicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37°C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering. PMID:20056274

  16. Enigmatic insight into collagen.

    PubMed

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  17. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  18. Deletion of the Small RNA Chaperone Protein Hfq down Regulates Genes Related to Virulence and Confers Protection against Wild-Type Brucella Challenge in Mice

    PubMed Central

    Lei, Shuangshuang; Zhong, Zhijun; Ke, Yuehua; Yang, Mingjuan; Xu, Xiaoyang; Ren, Hang; An, Chang; Yuan, Jiuyun; Yu, Jiuxuan; Xu, Jie; Qiu, Yefeng; Shi, Yanchun; Wang, Yufei; Peng, Guangneng; Chen, Zeliang

    2016-01-01

    Brucellosis is one of the most common zoonotic epidemics worldwide. Brucella, the etiological pathogen of brucellosis, has unique virulence characteristics, including the ability to survive within the host cell. Hfq is a bacterial chaperone protein that is involved in the survival of the pathogen under stress conditions. Moreover, hfq affects the expression of a large number of target genes. In the present study, we characterized the expression and regulatory patterns of the target genes of Hfq during brucellosis. The results revealed that hfq expression is highly induced in macrophages at the early infection stage and at the late stage of mouse infection. Several genes related to virulence, including omp25, omp31, vjbR, htrA, gntR, and dnaK, were found to be regulated by hfq during infection in BALB/c mice. Gene expression and cytokine secretion analysis revealed that an hfq-deletion mutant induced different cytokine profiles compared with that induced by 16M. Infection with the hfq-deletion mutant induced protective immune responses against 16M challenge. Together, these results suggest that hfq is induced during infection and its deletion results in significant attenuation which affects the host immune response caused by Brucella infection. By regulating genes related to virulence, hfq promotes the virulence of Brucella. The unique characteristics of the hfq-deletion mutant, including its decreased virulence and the ability to induce protective immune response upon infection, suggest that it represents an attractive candidate for the design of a live attenuated vaccine against Brucella. PMID:26834720

  19. Masculinisation of the Zebra Finch Song System: Roles of Oestradiol and the Z-chromosome Gene Tubulin-Specific Chaperone Protein A

    PubMed Central

    Beach, L. Q.; Wade, J.

    2015-01-01

    Robust sex differences in brain and behaviour exist in zebra finches. Only males sing, and forebrain song control regions are more developed in males. The factors driving these differences are not clear, although numerous experiments have shown that oestradiol (E2) administered to female hatchlings partially masculinises brain and behaviour. Recent studies suggest that an increased expression of Z-chromosome genes in males (ZZ; females: ZW) might also play a role. The Z-gene tubulin-specific chaperone A (TBCA) exhibits increased expression in the lateral magnocellular nucleus of the anterior nidopallium (LMAN) of juvenile males compared to females; TBCA+ cells project to the robust nucleus of the arcopallium (RA). In the present study, we investigated the role of TBCA and tested hypotheses with respect to the interactive or additive effects of E2 and TBCA. We first examined whether E2 in hatchling zebra finches modulates TBCA expression in the LMAN. It affected neither the mRNA, nor protein in either sex. We then unilaterally delivered TBCA small interfering (si)RNA to the LMAN of developing females treated with E2 or vehicle and males treated with the aromatase inhibitor, fadrozole, or its control. In both sexes, decreasing TBCA in LMAN reduced RA cell number, cell size and volume. It also decreased LMAN volume in females. Fadrozole in males increased LMAN volume and RA cell size. TBCA siRNA delivered to the LMAN also decreased the projection from this brain region to the RA, as indicated by anterograde tract tracing. The results suggest that TBCA is involved in masculinising the song system. However, because no interactions between the siRNA and hormone manipulations were detected, TBCA does not appear to modulate effects of E2 in the zebra finch song circuit. PMID:25702708

  20. Collagen I and the fibroblast: high protein expression requires a new paradigm of post-transcriptional, feedback regulation

    PubMed Central

    Schwarz, Richard I.

    2016-01-01

    Background Scaling protein production seems like a simple perturbation of transcriptional control. However, when embryonic tendon fibroblasts have to produce >50% procollagen and secrete it from the cell 4 times faster than the average protein, this taxes the cellular machinery and requires a fresh look at how the pathway is controlled. Ascorbate, a reducing agent, can stimulate procollagen production 6-fold. Procollagen mRNA levels goes up 6-fold but requires 3 days for the cell to accomplish this task. Secretion rates, the last cellular step in the process, also goes up 6-fold but this occurs in <1 h. What regulatory scheme is consistent with these properties? Scope of this review This review focuses on fibroblasts that make high levels of procollagen (type I) and how they regulate the collagen pathway. Data from many different labs are relevant to this problem but it is hard to see the bigger picture from a large number of small studies. This review aims to consolidate this data into a coherent model and this requires solutions to some controversies and postulating potential mechanisms where the details are still missing. Major Conclusions In high collagen producing cells, the pathway is controlled by post-transcriptional regulation. This requires feedback control between secretion and translation rates that is based on the helical structure of the procollagen molecule and additional tissue-specific modifications. General Significance Transcriptional control does not scale well to high protein production with rapid regulation. New paradigms lead to better understanding of collagen diseases and tendon morphogenesis. PMID:26900604

  1. MicroRNA-511 Binds to FKBP5 mRNA, Which Encodes a Chaperone Protein, and Regulates Neuronal Differentiation.

    PubMed

    Zheng, Dali; Sabbagh, Jonathan J; Blair, Laura J; Darling, April L; Wen, Xiaoqi; Dickey, Chad A

    2016-08-19

    Single nucleotide polymorphisms in the FKBP5 gene increase the expression of the FKBP51 protein and have been associated with increased risk for neuropsychiatric disorders such as major depression and post-traumatic stress disorder. Moreover, levels of FKBP51 are increased with aging and in Alzheimer disease, potentially contributing to disease pathogenesis. However, aside from its glucocorticoid responsiveness, little is known about what regulates FKBP5 In recent years, non-coding RNAs, and in particular microRNAs, have been shown to modulate disease-related genes and processes. The current study sought to investigate which miRNAs could target and functionally regulate FKBP5 Following in silico data mining and initial target expression validation, miR-511 was found to suppress FKBP5 mRNA and protein levels. Using luciferase p-miR-Report constructs and RNA pulldown assays, we confirmed that miR-511 bound directly to the 3'-UTR of FKBP5, validating the predicted gene-microRNA interaction. miR-511 suppressed glucocorticoid-induced up-regulation of FKBP51 in cells and primary neurons, demonstrating functional, disease-relevant control of the protein. Consistent with a regulator of FKBP5, miR-511 expression in the mouse brain decreased with age but increased following chronic glucocorticoid treatment. Analysis of the predicted target genes of miR-511 revealed that neurogenesis, neuronal development, and neuronal differentiation were likely controlled by these genes. Accordingly, miR-511 increased neuronal differentiation in cells and enhanced neuronal development in primary neurons. Collectively, these findings show that miR-511 is a functional regulator of FKBP5 and can contribute to neuronal differentiation. PMID:27334923

  2. Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation.

    PubMed

    Shu, Qin; Krezel, Andrzej M; Cusumano, Zachary T; Pinkner, Jerome S; Klein, Roger; Hultgren, Scott J; Frieden, Carl

    2016-06-28

    Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation. PMID:27298344

  3. Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

    PubMed

    Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

    2013-12-14

    Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. PMID:23890721

  4. Non-collagenous protein screening in the human chondrodysplasias: link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin.

    PubMed

    Stanescu, V; Do, T P; Chaminade, F; Maroteaux, P; Stanescu, R

    1994-05-15

    A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudoachondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia. PMID:8030664

  5. Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

    PubMed Central

    Sato, Koichi; Ishiai, Masamichi; Toda, Kazue; Furukoshi, Satoshi; Osakabe, Akihisa; Tachiwana, Hiroaki; Takizawa, Yoshimasa; Kagawa, Wataru; Kitao, Hiroyuki; Dohmae, Naoshi; Obuse, Chikashi; Kimura, Hiroshi; Takata, Minoru; Kurumizaka, Hitoshi

    2012-01-01

    Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair. PMID:22828868

  6. Mitotic regulator Mis18β interacts with and specifies the centromeric assembly of molecular chaperone holliday junction recognition protein (HJURP).

    PubMed

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-03-21

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437-460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  7. Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)*

    PubMed Central

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-01-01

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437–460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  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. Evidence for the presence of collagenous domains in Candida albicans cell surface proteins.

    PubMed Central

    Sepúlveda, P; Murgui, A; López-Ribot, J L; Casanova, M; Timoneda, J; Martínez, J P

    1995-01-01

    Rabbit polyclonal antibodies (PAbs) directed towards the amino-terminal cysteine-rich 7S domain (PAb anti-7S), the major internal collagenous domain (PAb anti-type IV), and the C-terminal noncollagenous region (PAb anti-NC1) of the type IV collagen molecule were probed by indirect immunofluorescence against Candida albicans blastoconidia and germinated blastoconidia. Most nongerminating cells and mother blastoconidia from which germ tubes originated showed strong fluorescence when PAb anti-7S was used, whereas with PAb anti-type IV, fluorescence was found almost exclusively on the surface of filamentous forms. A patched fluorescent pattern rather than a homogenous confluent fluorescence was observed in all cases. No fluorescent cells were observed with PAb anti-NC1. By Western immunoblotting, PAb anti-type IV cross-reacted primarily with a polypeptide of 37 kDa present in wall extracts obtained from intact cells of both growth forms by treatment with beta-mercaptoethanol, whereas PAb anti-7S recognized a major 58-kDa antigen also present in both extracts, along with some other high-molecular-mass (> 106-kDa) polydisperse species present only in the material released from blastoconidia with beta-mercaptoethanol. No reactive bands were observed when PAb anti-NC1 was used as a probe in Western immunoblotting experiments. The sensitivities or resistances to collagenase digestion of the different polypeptides that cross-reacted with PAbs anti-type IV and anti-7S suggest the existence of cell wall components in C. albicans that contain epitopes that mimic the collagenous domains of the type IV collagen molecule. PMID:7768595

  10. 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

  11. Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding

    PubMed Central

    Riordan, John R.; Dokholyan, Nikolay V.

    2008-01-01

    The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential

  12. Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.

    PubMed

    Serohijos, Adrian W R; Hegedus, Tamás; Riordan, John R; Dokholyan, Nikolay V

    2008-02-01

    The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-DeltaF508

  13. Collagen and gelatin.

    PubMed

    Liu, Dasong; Nikoo, Mehdi; Boran, Gökhan; Zhou, Peng; Regenstein, Joe M

    2015-01-01

    Collagen and gelatin have been widely used in the food, pharmaceutical, and cosmetic industries due to their excellent biocompatibility, easy biodegradability, and weak antigenicity. Fish collagen and gelatin are of renewed interest, owing to the safety and religious concerns of their mammalian counterparts. The structure of collagen has been studied using various modern technologies, and interpretation of the raw data should be done with caution. The structure of collagen may vary with sources and seasons, which may affect its applications and optimal extraction conditions. Numerous studies have investigated the bioactivities and biological effects of collagen, gelatin, and their hydrolysis peptides, using both in vitro and in vivo assay models. In addition to their established nutritional value as a protein source, collagen and collagen-derived products may exert various potential biological activities on cells in the extracellular matrix through the corresponding food-derived peptides after ingestion, and this might justify their applications in dietary supplements and pharmaceutical preparations. Moreover, an increasing number of novel applications have been found for collagen and gelatin. Therefore, this review covers the current understanding of the structure, bioactivities, and biological effects of collagen, gelatin, and gelatin hydrolysates as well as their most recent applications. PMID:25884286

  14. The Collagen-like Protein gp12 Is a Temperature-dependent Reversible Binder of SPP1 Viral Capsids*

    PubMed Central

    Zairi, Mohamed; Stiege, Asita C.; Nhiri, Naima; Jacquet, Eric; Tavares, Paulo

    2014-01-01

    Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface. PMID:25074929

  15. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    PubMed

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently. PMID:27293017

  16. COLLAGEN PROCESSING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Collagen dispersions, produced from fibrils recovered from milled bovine collagen, have shown promise in environmental remediation in applications as settling aids, filtration aids, fractionation media, oil drop stabilizers, and water purification aids. Macroporous structures, processed by controll...

  17. Oxidative damage to collagen.

    PubMed

    Monboisse, J C; Borel, J P

    1992-01-01

    Extracellular matrix molecules, such as collagens, are good targets for oxygen free radicals. Collagen is the only protein susceptible to fragmentation by superoxide anion as demonstrated by the liberation of small 4-hydroxyproline-containing-peptides. It seems likely that hydroxyl radicals in the presence of oxygen cleave collagen into small peptides, and the cleavage seems to be specific to proline or 4-hydroxyproline residues. Hydroxyl radicals in the absence of oxygen or hypochlorous acid do not induce fragmentation of collagen molecules, but they trigger a polymerization of collagen through the formation of new cross-links such as dityrosine or disulfure bridges. Moreover, these cross-links can not explain the totality of high molecular weight components generated under these experimental conditions, and the nature of new cross-links induced by hydroxyl radicals or hypochlorous acid remains unclear. PMID:1333311

  18. Protein kinase signalling pathways involved in the up-regulation of the rat alpha1(I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells.

    PubMed Central

    Palcy, S; Goltzman, D

    1999-01-01

    Transforming growth factor beta (TGFbeta) family members are known for their important role in bone physiology. TGFbeta(1) and, to a smaller extent, bone morphogenetic protein 2 (BMP-2) have been reported to regulate the gene expression of different osteoblast markers in vitro. However, little is known about the molecular mechanisms involved in these actions. Here we report that BMP-2, like TGFbeta(1), up-regulated alpha1(I) collagen mRNA expression in ROS 17/2.8 osteoblastic cells. This was mediated through an increase in the transcriptional rate of the gene rather than through the stabilization of alpha1(I) collagen mRNA, and required new protein synthesis. In addition, TGFbeta(1)- and BMP-2-induced increases in alpha1(I) collagen mRNA levels were both dependent on protein kinase C and protein tyrosine kinase activities. Furthermore, the mitogen-activated protein kinase (MAPK) [MAPK/extracellular signal-regulated protein kinase kinase 1/extracellular signal-regulated protein kinase (MEK-1/ERK)] pathway participated in the up-regulation of alpha1(I) collagen gene expression by TGFbeta(1) and BMP-2. In response to either TGFbeta(1) or BMP-2, the stimulation of alpha1(I) collagen mRNA levels was paralleled by an early increase in extracellular signal-regulated kinase protein activity. Moreover, the effects of both TGFbeta(1) and BMP-2 on alpha1(I) collagen gene expression were markedly decreased in transfected ROS 17/2.8 cells expressing a dominant-negative MEK-1. Our findings therefore show that TGFbeta(1) and BMP-2, which signal through discrete cell-surface receptors, are able to trigger analogous, if not identical, protein-phosphorylation-transducing cascades leading to comparable actions on the transcription of the alpha1(I) collagen gene in osteoblastic cells. PMID:10493907

  19. 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

  20. Increased expression of platelet-derived growth factor A and collagenous matrix proteins in congenital multicystic renal dysplasia.

    PubMed

    Liapis, H; Yu, H; Flath, A; Steinhardt, G F

    1997-01-01

    The expression of platelet-derived growth factor A (PDGF-A), and its spatial and temporal relationship to interstitial collagens in kidneys with congenital multicystic dysplasia using in situ hybridization, have been examined. Seventeen dysplastic kidneys (16 weeks to 7 months) and 20 normal age-matched controls were used in the study. Increased PDGF-A mRNA was detected in dysplastic compared to normal kidneys in all age groups including extensively fibrotic postnatal kidneys. An abundant PDGF-A mRNA signal was seen within the epithelial cells of cystically dilated or dysplastic tubules and within interstitial fibroblasts and disorganized primitive mesenchyme. A comparable amount of PDGF-A protein was detected by Western blotting. Procollagen I and III mRNA were increased in fibroblasts surrounding cystic and dysplastic tubules. We conclude that tubular epithelial production of PDGF-A may induce collagenous matrix production by adjacent fibroblasts, while marked up-regulation of PDGF-A by interstitial cells may be responsible for sustainable fibrogenic effects in the fetal kidney contributing to renal maldevelopment. PMID:9200407

  1. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    NASA Astrophysics Data System (ADS)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  2. Hydration properties of the molecular chaperone alpha-crystallin in the bovine lens.

    PubMed

    Babizhayev, M A; Nikolayev, G M; Goryachev, S N; Bours, J; Martin, R

    2003-10-01

    Topographic studies of crystalline fractions from different morphological layers of the young adult bovine lens were conducted. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel (IEF). Water soluble (WS) crystallins from the lens equator revealed a separation into HM (high molecular weight) alpha(L)-, beta(H)-, beta(L)-, beta(S)-, and gamma-crystallins. The nature of the water insoluble (WI) protein fraction in the separated lens layers reflected the aggregated state of alpha(L)-, beta(L)-, beta(S)-, and gamma-crystallins in different regions of the lens, concealed in the central cavity of the alpha-crystallin chaperone model. The IEF data demonstrate a possible chaperone-like function for alpha-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. The water binding properties of bovine lens alpha-crystallin, calf skin collagen, and bovine serum albumin (BSA) were investigated with various techniques. The water adsorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The NMR spin-echo technique was used to study the hydration of protein samples and to determine the spin-spin relaxation times (T(2)) from the protons of water adsorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During water vapor uptake at relative humidity P/P(0) = 0.75, alpha-crystallin did not adsorb water suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At relative humidity P/P(0) = 1.0, the adsorption of water by alpha-crystallin was 17% with a single component decay character of spin echo (T(2) = 3 msec). Addition of water to alpha-crystallin to about 50% of its

  3. WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability.

    PubMed

    Ham, Jong Hyun; Majerczak, Doris R; Arroyo-Rodriguez, Angel S; Mackey, David M; Coplin, David L

    2006-10-01

    The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system. PMID:17022173

  4. Heat Shock Protein gp96 Is a Master Chaperone for Toll-like Receptors and Is Important in the Innate Function of Macrophages

    PubMed Central

    Yang, Yi; Liu, Bei; Dai, Jie; Srivastava, Pramod K.; Zammit, David J.; Lefrançois, Leo; Li, Zihai

    2010-01-01

    SUMMARY gp96 is an endoplasmic reticulum chaperone for cell-surface Toll-like receptors (TLRs). Little is known about its roles in chaperoning other TLRs or in the biology of macrophage in vivo. We generated a macrophage-specific gp96-deficient mouse. Despite normal development and activation by interferon-γ, tumor necrosis factor-α, and interleukin-1β, the mutant macrophages failed to respond to ligands of both cell-surface and intracellular TLRs including TLR2, TLR4, TLR5, TLR7, and TLR9. Furthermore, we found that TLR4 and TLR9 preferentially interacted with a super-glycosylated gp96 species. The categorical loss of TLRs in gp96-deficient macrophages operationally created a conditional and cell-specific TLR null mouse. These mice were resistant to endotoxin shock but were highly susceptible to Listeria monocytogenes. Our results demonstrate that gp96 is the master chaperone for TLRs and that macrophages, but not other myeloid cells, are the dominant source of proinflammatory cytokines during endotoxemia and Listeria infections. PMID:17275357

  5. [Acid-soluble collagen in the skin of rats who received a tryptophan load and were kept on a protein-free diet].

    PubMed

    Pechenova, T N; Gulyĭ, M F; Volodina, T T; Solodova, E V; Popova, N N

    1983-01-01

    Crystalline preparations of acid-soluble collagen of rat skin in norm and with tryptophan load against a background of the protein-free diet were fractionated by differential salting out using NaCl in concentrations corresponding to precipitation zones of collagen 1,3 and 4. Amino acid composition, electrophoretic mobility in polyacrylamide gel, profiles of elution from CM-cellulose, content of the carbohydrate component and--S--S-bonds were studied in proteins of the mentioned fractions and in the precipitate insoluble after the pepsin action. Essential differences as compared to the normal level in the amino acid composition and elution profiles were detected in the fraction corresponding to collagen I. Quantitative changes in the carbohydrate component and--S--S-bonds occur due to the fraction inaccessible to the persin action. PMID:6829075

  6. NMR spin-echo studies of hydration properties of the molecular chaperone alpha-crystallin in the bovine lens.

    PubMed

    Babizhayev, Mark A; Nikolayev, Gennady N; Goryachev, Sergey N; Bours, Johan

    2002-07-29

    The water-binding properties of bovine lens alpha-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin-echo technique was used to study the hydration of protein samples and to determine the spin-spin relaxation times (T2) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P(0)=0.75, alpha-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P(0)=1.0, the absorption of water by alpha-crystallin was 17% with a single component decay character of spin-echo (T2=3 ms). Addition of water to alpha-crystallin to about 50% of its w/w in the protein sample showed T2=8 ms with only one single component decay of the spin-echo signal. The single component decay character of the spin-echo indicates at the tightly bound water by alpha-crystallin. Under a relative humidity P/P(0)=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T2 of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with alpha-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for alpha-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To

  7. Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease

    PubMed Central

    Daans, Melina; Lories, Rik JU; Luyten, Frank P

    2008-01-01

    Introduction Rheumatoid arthritis is a chronic systemic autoimmune disease affecting peripheral joints and leading to loss of joint function. The severity and outcome of disease are dependent on the balance between inflammatory/destructive and homeostatic or repair pathways. Increasing evidence suggests a role for bone morphogenetic protein (BMP) signaling in joint homeostasis and disease. Methods Activation of BMP signaling in collagen-induced arthritis as a model of rheumatoid arthritis was studied by immunohistochemistry and Western blot for phosphorylated SMAD1/5 at different time points. Expression of different BMP ligands and noggin, a BMP antagonist, was determined on synovium and cartilage extracts of arthritic knees, at different time points, with quantitative polymerase chain reaction. At the protein level, BMP2 and BMP7 were studied with immunohistochemistry. Finally, the effect of anti-tumor necrosis factor-alpha (TNFα) treatment on the expression of BMP2, BMP7, and growth and differentiation factor-5 (GDF5) in synovium and cartilage of arthritic knees was investigated. Results A time-dependent activation of the BMP signaling pathway in collagen-induced arthritis was demonstrated with a dynamic and characteristic expression pattern of different BMP subfamily members in synovium and cartilage of arthritic knees. As severity increases, the activation of BMP signaling becomes more prominent in the invasive pannus tissue. BMP2 is present in cartilage and the hyperplastic lining layer. BMP7 is found in the sublining zone and inflammatory infiltrate. Treatment with etanercept slowed down progression of disease, but no change in expression of GDF5, BMP2, and BMP7 in synovium was found; in the cartilage, however, blocking of TNFα increased the expression of BMP7. Conclusions BMP signaling is dynamically activated in collagen-induced arthritis and is partly TNFα-independent. TNFα blocking increased the expression of BMP7 in the articular cartilage, possibly

  8. Y-box proteins interact with the S1 nuclease-sensitive site in the chicken alpha 2(I) collagen gene promoter.

    PubMed Central

    Bayarsaihan, D; Enkhmandakh, B; Lukens, L N

    1996-01-01

    The sequence of the chicken alpha 2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293-296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken alpha 2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes. PMID:8870670

  9. Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

    PubMed Central

    Gao, Peng; Pinkston, Kenneth L.; Bourgogne, Agathe; Cruz, Melissa R.; Garsin, Danielle A.; Murray, Barbara E.

    2013-01-01

    The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. PMID:23974022

  10. 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

  11. 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

  12. 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

  13. 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

  14. Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa.

    PubMed

    Discola, Karen F; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-02-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  15. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  16. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

    PubMed Central

    Agueli, C; Geraci, F; Giudice, G; Chimenti, L; Cascino, D; Sconzo, G

    2001-01-01

    In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters. PMID:11716770

  17. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

    PubMed

    Agueli, C; Geraci, F; Giudice, G; Chimenti, L; Cascino, D; Sconzo, G

    2001-12-01

    In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters. PMID:11716770

  18. The new flagella-associated collagen-like proteins ClpB and ClpC of Bacillus amyloliquefaciens FZB42 are involved in bacterial motility.

    PubMed

    Zhao, Xia; Wang, Ruoyu; Shang, Qianhan; Hao, Haiting; Li, Yuyao; Zhang, Yubao; Guo, Zhihong; Wang, Yun; Xie, Zhongkui

    2016-03-01

    Collagen-like proteins (CLPs) share the distinctive Gly-X-Thr repeating amino acid sequence of animal collagens, and contain N- and C-terminal domain making a collagen-like structure in Bacillus amyloliquefaciens FZB42, a plant growth-promoting rhizobacterium. Our previous study demonstrated that CLPs play important roles in biofilm construction and adherence to the surfaces on plant roots. However, bacterial localization of the CLPs remains unclear. Here, disrupted strains on all four clp genes (clpA, clpB, clpC and clpD) shown fewer filament than wild-type bacteria in extracellular matrix under scanning electron microscope (SEM). Transmission electron microscopy (TEM) was used to observe the differences on filament which associated on the cell surface, then the CLPs mutation strains showed less flagella than the wild type. Immunogold labeling determined the location that ClpB and ClpC localized on the flagella surface. In addition, western blotting analysis of crude flagella extracts suggested that the ClpB and ClpC are associated to flagella as well. The mutation strains also reduced motility of swimming on the surface of soft agar medium and changed the architectural of microcolony biofilm edge. The study suggests that collagen-like protein ClpB and ClpC, as novel proteins, associated with flagella in B. amyloliquefaciens. PMID:26856450

  19. RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

    PubMed Central

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-01-01

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  20. RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism.

    PubMed

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-12-23

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  1. Contribution of the Collagen-Binding Proteins of Streptococcus mutans to Bacterial Colonization of Inflamed Dental Pulp

    PubMed Central

    Nomura, Ryota; Ogaya, Yuko; Nakano, Kazuhiko

    2016-01-01

    Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases. PMID:27442266

  2. 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

  3. 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.

  4. 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

  5. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    SciTech Connect

    Choudhury, Kamalika Roy; Bhattacharyya, Nitai P.

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  6. The efficiency of magnetic hyperthermia and in vivo histocompatibility for human-like collagen protein-coated magnetic nanoparticles

    PubMed Central

    Chang, Le; Liu, Xiao Li; Di Fan, Dai; Miao, Yu Qing; Zhang, Huan; Ma, He Ping; Liu, Qiu Ying; Ma, Pei; Xue, Wei Ming; Luo, Yan E; Fan, Hai Ming

    2016-01-01

    Magnetic hyperthermia is a promising technique for the minimally invasive elimination of solid tumors. In this study, uniform magnetite nanoparticles (MNPs) with different particle sizes were used as a model system to investigate the size and surface effects of human-like collagen protein-coated MNPs (HLC-MNPs) on specific absorption rate and biocompatibility. It was found that these HLC-MNPs possess rapid heating capacity upon alternating magnetic field exposure compared to that of MNPs without HLC coating, irrespective of the size of MNPs. The significant enhancement of specific absorption rate is favorable for larger sized nanoparticles. Such behavior is attributed to the reduced aggregation and increased stability of the HLC-MNPs. By coating HLC on the surface of certain sized MNPs, a significant increase in cell viability (up to 2.5-fold) can be achieved. After subcutaneous injection of HLC-MNPs into the back of Kunming mice, it was observed that the inflammatory reaction hardly occurred in the injection site. However, there was a significant presence of phagocytes and endocytosis after the injection of nonconjugated counterparts. The overall strategy to fabricate HLC-MNPs can serve as a general guideline to address the current challenges in clinical magnetic hyperthermia, improved biocompatibility, and enhanced heating characteristics through protein coating. PMID:27042065

  7. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein.

    PubMed

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5'-untranslated region (5'-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic "on" switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  8. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

    PubMed Central

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5′-untranslated region (5′-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic “on” switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  9. 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

  10. 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

  11. Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation.

    PubMed

    Fruchtl, McKinzie; Sakon, Joshua; Beitle, Robert

    2016-05-20

    The production of collagen binding domain fusion proteins is of significant importance because of their potential as therapeutic biomaterials. It was previously reported that the expression of collagen-binding domain fusion proteins in Escherichia coli was higher when expressed using lactose as an inducer and chemically defined growth media on a shake flask scale. In an effort to further investigate factors that affect expression levels on a fed-batch scale, alternative induction techniques were tested in conjunction with fed-batch fermentation. In this paper, we discuss ten fed-batch fermentation experiments utilizing either glucose or glycerol feed and using lactose or isopropyl-β-d-thiogalactopyranoside (IPTG) as an induction source. It was found that glycerol-fed fermentations induced with lactose allowed for greater expression of target protein, though lesser cell densities were achieved. PMID:26975843

  12. Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif.

    PubMed

    Parish, David; Benach, Jordi; Liu, Goahua; Singarapu, Kiran Kumar; Xiao, Rong; Acton, Thomas; Su, Min; Bansal, Sonal; Prestegard, James H; Hunt, John; Montelione, Gaetano T; Szyperski, Thomas

    2008-12-01

    The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides. PMID:19039680

  13. Protein Chaperones Q8ZP25_SALTY from Salmonella Typhimurium and HYAE_ECOLI from Escherichia coli Exhibit Thioredoxin-like Structures Despite Lack of Canonical Thioredoxin Active Site Sequence Motif

    SciTech Connect

    Parish, D.; Benach, J; Liu, G; Singarapu, K; Xiao, R; Acton, T; Hunt, J; Montelione, G; Szyperski, T; et. al.

    2008-01-01

    The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe) hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

  14. The C. elegans UNC-23 protein, a member of the BCL-2-associated athanogene (BAG) family of chaperone regulators, interacts with HSP-1 to regulate cell attachment and maintain hypodermal integrity

    PubMed Central

    Rahmani, Poupak; Rogalski, Teresa; Moerman, Donald G

    2015-01-01

    Mutations in the unc-23 gene in the free-living nematode, Caenorhabditis elegans result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate. We have determined that the unc-23 gene product is the nematode ortholog of the human BAG-2 protein, a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. We show that a functional GFP-tagged UNC-23 protein is expressed throughout development in several tissues of the animal, including body wall muscle and hypodermis, and associates with adhesion complexes and attachment structures within these 2 tissues. In humans, the BAG protein family consists of 6 members that all contain a conserved 45 amino acid BAG domain near their C-termini. These proteins bind to and modulate the activity of the ATPase domain of the heat shock cognate protein 70, Hsc70. We have isolated missense mutations in the ATPase domain of the C. elegans heat shock 70 protein, HSP-1 that suppress the phenotype exhibited by unc-23(e25) mutant hermaphrodites and we show that UNC-23 and HSP-1 interact in a yeast-2-hybrid system. The interaction of UNC-23 with HSP-1 defines a role for HSP-1 function in the maintenance of muscle attachment during development. PMID:26435886

  15. 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

  16. 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

  17. 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

  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. A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

    PubMed Central

    Borrero-de Acuña, José Manuel; Molinari, Gabriella; Rohde, Manfred; Dammeyer, Thorben; Wissing, Josef; Jänsch, Lothar; Arias, Sagrario; Jahn, Martina; Schobert, Max; Timmis, Kenneth N.

    2015-01-01

    ABSTRACT Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCE Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm

  20. Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

    PubMed

    Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

    2016-04-29

    Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

  1. 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

  2. Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

    PubMed Central

    Mann, K; Deutzmann, R; Aumailley, M; Timpl, R; Raimondi, L; Yamada, Y; Pan, T C; Conway, D; Chu, M L

    1989-01-01

    The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized. Images PMID:2496973

  3. Co-delivery and controlled release of stromal cell-derived factor-1α chemically conjugated on collagen scaffolds enhances bone morphogenetic protein-2-driven osteogenesis in rats

    PubMed Central

    SUN, HAIPENG; WANG, JINMING; DENG, FEILONG; LIU, YUN; ZHUANG, XIUMEI; XU, JIAYUN; LI, LONG

    2016-01-01

    There has been considerable focus in investigations on the delivery systems and clinical applications of bone morphogenetic protein-2 (BMP-2) for novel bone formation. However, current delivery systems require high levels of BMP-2 to exert a biological function. There are several concerns in using of high levels of BMP-2, including safety and the high cost of treatment. Therefore, the development of strategies to decrease the levels of BMP-2 required in these delivery systems is required. In our previous studies, a controlled-release system was developed, which used Traut's reagent and the cross-linker, 4-(N-maleimi-domethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), to chemically conjugate BMP-2 directly on collagen discs. In the current study, retention efficiency and release kinetics of stromal cell-derived factor-1α (SDF-1α) cross-linked on collagen scaffolds were detected. In addition, the osteogenic activity of SDF-1α and suboptimal doses of BMP-2 cross-linked on collagen discs following subcutaneous implantation in rats were evaluated. Independent two-tailed t-tests and one-way analysis of variance were used for analysis. In the present study, the controlled release of SDF-1α chemically conjugated on collagen scaffolds was demonstrated. By optimizing the concentrations of Traut's reagent and the Sulfo-SMCC cross-linker, a significantly higher level of SDF-1α was covalently retained on the collagen scaffold, compared with that retained using a physical adsorption method. Mesenchymal stem cell homing indicated that the biological function of the SDF-1α cross-linked on the collagen scaffolds remained intact. In rats, co-treatment with SDF-1α and a suboptimal dose of BMP-2 cross-linked on collagen scaffolds using this chemically conjugated method induced higher levels of ectopic bone formation, compared with the physical adsorption method. No ectopic bone formation was observed following treatment with a

  4. Exposure to Mimivirus Collagen Promotes Arthritis

    PubMed Central

    Shah, Nikunj; Hülsmeier, Andreas J.; Hochhold, Nina; Neidhart, Michel; Gay, Steffen

    2014-01-01

    Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localiza