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Sample records for protein functions upstream

  1. In vivo "photofootprint" changes at sequences between the yeast GAL1 upstream activating sequence and "TATA" element require activated GAL4 protein but not a functional TATA element.

    PubMed Central

    Selleck, S B; Majors, J

    1988-01-01

    Transcription of the yeast GAL1 and GAL10 genes is induced by growth on galactose. Using the technique of photofootprinting in vivo, we previously documented equivalent transcription-dependent footprints within the putative "TATA" elements of both genes. To explore the functional significance of these observations, we created a 3-base-pair substitution mutation within the GAL1 promoter TATA element, which disrupted the ATATAA consensus sequence but left intact the photomodification targets. The mutation reduced galactose-induced RNA levels by a factor of 100. The mutant promoter no longer displayed the characteristic TATA sequence footprint, supporting the hypothesis that transcription activation involves the binding of a TATA box factor. We also observed a collection of transcription-correlated alterations in the modification pattern at sites between the UASG and the GAL1 TATA element, within sequences that are not required for inducible transcription. These patterns, characteristic of the induced wild-type GAL1 gene, were still galactose inducible with the TATA mutant GAl1 promoter, despite the low level of transcription from this promoter. We conclude that the GAL4-dependent protein/DNA structure responsible for the altered pattern within nonessential sequences is therefore not strictly coupled to an active TATA element or to high levels of expression. Nonetheless, the patterns probably reflect a stable protein-dependent structure that accompanies assembly of the transcription initiation complex. Images PMID:3041409

  2. Upstream Pathways Controlling Mitochondrial Function in Major Psychosis

    PubMed Central

    Machado, Alencar Kolinski; Pan, Alexander Yongshuai; da Silva, Tatiane Morgana; Duong, Angela

    2016-01-01

    Mitochondrial dysfunction is commonly observed in bipolar disorder (BD) and schizophrenia (SCZ) and may be a central feature of psychosis. These illnesses are complex and heterogeneous, which is reflected by the complexity of the processes regulating mitochondrial function. Mitochondria are typically associated with energy production; however, dysfunction of mitochondria affects not only energy production but also vital cellular processes, including the formation of reactive oxygen species, cell cycle and survival, intracellular Ca2+ homeostasis, and neurotransmission. In this review, we characterize the upstream components controlling mitochondrial function, including 1) mutations in nuclear and mitochondrial DNA, 2) mitochondrial dynamics, and 3) intracellular Ca2+ homeostasis. Characterizing and understanding the upstream factors that regulate mitochondrial function is essential to understand progression of these illnesses and develop biomarkers and therapeutics. PMID:27310240

  3. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    SciTech Connect

    Yoshimura, Akari; Kobayashi, Yume; Tada, Shusuke; Seki, Masayuki; Enomoto, Takemi

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  4. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  5. Translational control of protein kinase Ceta by two upstream open reading frames.

    PubMed

    Raveh-Amit, Hadas; Maissel, Adva; Poller, Jonathan; Marom, Liraz; Elroy-Stein, Orna; Shapira, Michal; Livneh, Etta

    2009-11-01

    Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKCeta, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5' untranslated region (5' UTR) of PKCeta is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5' UTR and show their effect in suppressing the expression of PKCeta in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKCeta in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2alpha phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.

  6. The functional response of upstream DNA to dynamic supercoiling in vivo.

    PubMed

    Kouzine, Fedor; Sanford, Suzanne; Elisha-Feil, Zichrini; Levens, David

    2008-02-01

    Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation.

  7. Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

    PubMed Central

    Ham, J; Dostatni, N; Arnos, F; Yaniv, M

    1991-01-01

    The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation. Images PMID:1655407

  8. Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation

    PubMed Central

    Ferreira, Joshua P; Noderer, William L; Diaz de Arce, Alexander J; Wang, Clifford L

    2014-01-01

    We have employed upstream open reading frames (uORFs) to systematically tune the translation levels of recombinant proteins. We present the design principles that guided the development of this technology and provide information that may help others in implementing synthetic uORFs for their own applications. We also report on recent applications to our own research projects, including the coupling of uORF and translation initiation site (TIS) engineering with small molecule-inducible post-translational control. Finally, we discuss opportunities to investigate and potentially engineer gene-specific translational responses to cellular stress. PMID:24637490

  9. Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

    PubMed Central

    Zhou, Weixin; Chung, Yang Jo; Parrilla Castellar, Edgardo R.; Zheng, Ying; Chung, Hye-Jung; Bandle, Russell; Liu, Juhong; Tessarollo, Lino; Batchelor, Eric; Aplan, Peter D.; Levens, David

    2017-01-01

    The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1−/−) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1−/− hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1−/− hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1−/− embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression. PMID:26774856

  10. Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation

    PubMed Central

    Bal, Natalia V.; Susorov, Denis; Chesnokova, Ekaterina; Kasianov, Artem; Mikhailova, Tatiana; Alkalaeva, Elena; Balaban, Pavel M.; Kolosov, Peter

    2016-01-01

    For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5′ untranslated region (5′UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKMζ translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKMζ leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKMζ 5′UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKMζ translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKMζ translation in activated neurons, important at early stages of LTP. PMID:27790092

  11. CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

    PubMed Central

    Wright, K L; Vilen, B J; Itoh-Lindstrom, Y; Moore, T L; Li, G; Criscitiello, M; Cogswell, P; Clarke, J B; Ting, J P

    1994-01-01

    NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element. Images PMID:8076600

  12. The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

    PubMed

    Wang, Ying; Yan, Jie; Lee, Haeryun; Lu, Qiuheng; Adler, Paul N

    2014-10-01

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

  13. Sonic hedgehog functions upstream of disrupted-in-schizophrenia 1 (disc1): implications for mental illness

    PubMed Central

    Boyd, Penelope J.; Cunliffe, Vincent T.; Roy, Sudipto; Wood, Jonathan D.

    2015-01-01

    ABSTRACT DISRUPTED-IN-SCHIZOPHRENIA (DISC1) has been one of the most intensively studied genetic risk factors for mental illness since it was discovered through positional mapping of a translocation breakpoint in a large Scottish family where a balanced chromosomal translocation was found to segregate with schizophrenia and affective disorders. While the evidence for it being central to disease pathogenesis in the original Scottish family is compelling, recent genome-wide association studies have not found evidence for common variants at the DISC1 locus being associated with schizophrenia in the wider population. It may therefore be the case that DISC1 provides an indication of biological pathways that are central to mental health issues and functional studies have shown that it functions in multiple signalling pathways. However, there is little information regarding factors that function upstream of DISC1 to regulate its expression and function. We herein demonstrate that Sonic hedgehog (Shh) signalling promotes expression of disc1 in the zebrafish brain. Expression of disc1 is lost in smoothened mutants that have a complete loss of Shh signal transduction, and elevated in patched mutants which have constitutive activation of Shh signalling. We previously demonstrated that disc1 knockdown has a dramatic effect on the specification of oligodendrocyte precursor cells (OPC) in the hindbrain and Shh signalling is known to be essential for the specification of these cells. We show that disc1 is prominently expressed in olig2-positive midline progenitor cells that are absent in smo mutants, while cyclopamine treatment blocks disc1 expression in these cells and mimics the effect of disc1 knock down on OPC specification. Various features of a number of psychiatric conditions could potentially arise through aberrant Hedgehog signalling. We therefore suggest that altered Shh signalling may be an important neurodevelopmental factor in the pathobiology of mental illness. PMID

  14. Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.

    PubMed

    Min, Cheng; Wenjian, Zhang; Tianyu, Xing; Xiaohong, Yan; Yumao, Li; Hui, Li; Ning, Wang

    2016-08-01

    miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19

  15. The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice

    PubMed Central

    Suharsono, Utut; Fujisawa, Yukiko; Kawasaki, Tsutomu; Iwasaki, Yukimoto; Satoh, Hikaru; Shimamoto, Ko

    2002-01-01

    We used rice dwarf1 (d1) mutants lacking a single-copy Gα gene and addressed Gα's role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H2O2 production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Gα mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Gα in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling. PMID:12237405

  16. Tying Down Loose Ends in the Chlamydomonas Genome: Functional Significance of Abundant Upstream Open Reading Frames

    PubMed Central

    Cross, Frederick R.

    2015-01-01

    The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. The annotated genome is very rich in open reading frames upstream of the annotated coding sequence (‘uORFs’): almost three quarters of the assigned transcripts have at least one uORF, and frequently more than one. This is problematic with respect to the standard ‘scanning’ model for eukaryotic translation initiation. These uORFs can be grouped into three classes: class 1, initiating in-frame with the coding sequence (CDS) (thus providing a potential in-frame N-terminal extension); class 2, initiating in the 5′ untranslated sequences (5UT) and terminating out-of-frame in the CDS; and class 3, initiating and terminating within the 5UT. Multiple bioinformatics criteria (including analysis of Kozak consensus sequence agreement and BLASTP comparisons to the closely related Volvox genome, and statistical comparison to cds and to random sequence controls) indicate that of ∼4000 class 1 uORFs, approximately half are likely in vivo translation initiation sites. The proposed resulting N-terminal extensions in many cases will sharply alter the predicted biochemical properties of the encoded proteins. These results suggest significant modifications in ∼2000 of the ∼20,000 transcript models with respect to translation initiation and encoded peptides. In contrast, class 2 uORFs may be subject to purifying selection, and the existent ones (surviving selection) are likely inefficiently translated. Class 3 uORFs are found in more than half of transcripts, frequently multiple times per transcript; however, they are remarkably similar to random sequence expectations with respect to size, number, and composition, and therefore may in most cases be selectively neutral. PMID:26701783

  17. Zinc Finger Protein5 Is Required for the Control of Trichome Initiation by Acting Upstream of Zinc Finger Protein8 in Arabidopsis1[C][W][OA

    PubMed Central

    Zhou, Zhongjing; An, Lijun; Sun, Lili; Zhu, Shuijin; Xi, Wanyan; Broun, Pierre; Yu, Hao; Gan, Yinbo

    2011-01-01

    Arabidopsis (Arabidopsis thaliana) trichome development is a model system for studying cell development, cell differentiation, and the cell cycle. Our previous studies have shown that the GLABROUS INFLORESCENCE STEMS (GIS) family genes, GIS, GIS2, and ZINC FINGER PROTEIN8 (ZFP8), control shoot maturation and epidermal cell fate by integrating gibberellins (GAs) and cytokinin signaling in Arabidopsis. Here, we show that a new C2H2 zinc finger protein, ZFP5, plays an important role in controlling trichome cell development through GA signaling. Overexpression of ZFP5 results in the formation of ectopic trichomes on carpels and other inflorescence organs. zfp5 loss-of-function mutants exhibit a reduced number of trichomes on sepals, cauline leaves, paraclades, and main inflorescence stems in comparison with wild-type plants. More importantly, it is found that ZFP5 mediates the regulation of trichome initiation by GAs. These results are consistent with ZFP5 expression patterns and the regional influence of GA on trichome initiation. The molecular analyses suggest that ZFP5 functions upstream of GIS, GIS2, ZFP8, and the key trichome initiation regulators GLABROUS1 (GL1) and GL3. Using a steroid-inducible activation of ZFP5 and chromatin immunoprecipitation experiments, we further demonstrate that ZFP8 is the direct target of ZFP5 in controlling epidermal cell differentiation. PMID:21803862

  18. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  19. [Cloning and function identification of gene 'admA' and up-stream regulatory sequence related to antagonistic activity of Enterobacter cloacae B8].

    PubMed

    Zhu, Jun-Li; Li, De-Bao; Yu, Xu-Ping

    2012-04-01

    To reveal the antagonistic mechanism of B8 strain to Xanthomonas oryzae pv. oryzae, transposon tagging method and chromosome walking were deployed to clone antagonistic related fragments around Tn5 insertion site in the mutant strain B8B. The function of up-stream regulatory sequence of gene 'admA' involved in the antagonistic activity was further identified by gene knocking out technique. An antagonistic related left fragment of Tn5 insertion site, 2 608 bp in length, was obtained by tagging with Kan resistance gene of Tn5. A 2 354 bp right fragment of Tn5 insertion site was amplified with 2 rounds of chromosome walking. The length of the B contig around the Tn5 insertion site was 4 611 bp, containing 7 open reading frames (ORFs). Bioinformatic analysis revealed that these ORFs corresponded to the partial coding regions of glyceraldehyde-3-phosphate dehydrogenase, two LysR family transcriptional regulators, hypothetical protein VSWAT3-20465 of Vibrionales and admA, admB, and partial sequence of admC gene of Pantoea agglomerans biosynthetic gene cluster, respectively. Tn5 was inserted in the up-stream of 200 bp or 894 bp of the sequence corresponding to anrP ORF or admA gene on B8B, respectively. The B-1 and B-2 mutants that lost antagonistic activity were selected by homeologuous recombination technology in association with knocking out plasmid pMB-BG. These results suggested that the transcription and expression of anrP gene might be disrupted as a result of the knocking out of up-stream regulatory sequence by Tn5 in B8B strain, further causing biosythesis regulation of the antagonistic related gene cluster. Thus, the antagonistic related genes in B8 strain is a gene family similar as andrimid biosynthetic gene cluster, and the upstream regulatory region appears to be critical for the antibiotics biosynthesis.

  20. Fusion activity of African henipavirus F proteins with a naturally occurring start codon directly upstream of the signal peptide.

    PubMed

    Weis, Michael; Behner, Laura; Binger, Tabea; Drexler, Jan Felix; Drosten, Christian; Maisner, Andrea

    2015-04-02

    Compared to the fusion proteins of pathogenic Nipah and Hendra viruses, the F protein of prototype African henipavirus GH-M74a displays a drastically reduced surface expression and fusion activity. A probable reason for limited F expression is the unusually long sequence located between the gene start and the signal peptide (SP) not present in other henipaviruses. Such a long pre-SP extension can prevent efficient ER translocation or protein maturation and processing. As its truncation can therefore enhance surface expression, the recent identification of a second in-frame start codon directly upstream of the SP in another African henipavirus F gene (GH-UP28) raised the question if such a naturally occurring minor sequence variation can lead to the synthesis of a pre-SP truncated translation product, thereby increasing the production of mature F proteins. To test this, we analyzed surface expression and biological activity of F genes carrying the second SP-proximal start codon of GH-UP28. Though we observed minor differences in the expression levels, introduction of the additional start codon did not result in an increased fusion activity, even if combined with further mutations in the pre-SP region. Thus, limited bioactivity of African henipavirus F protein is maintained even after sequence changes that alter the gene start allowing the production of F proteins without an unusually long pre-SP.

  1. Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

    PubMed

    Robert, Stéphanie; Goulet, Marie-Claire; D'Aoust, Marc-André; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

  2. A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation.

    PubMed Central

    Fashena, S J; Reeves, R; Ruddle, N H

    1992-01-01

    Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre-B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG-I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers. Images PMID:1732752

  3. Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

    PubMed

    Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

    2017-05-01

    Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

  4. PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis.

    PubMed Central

    Zhou, N; Tootle, T L; Tsui, F; Klessig, D F; Glazebrook, J

    1998-01-01

    The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented. PMID:9634589

  5. The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria.

    PubMed

    Sung, Tzu-Ying; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2010-08-01

    In Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from -1 to -15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

  6. The in Vivo TRPV6 Protein Starts at a Non-AUG Triplet, Decoded as Methionine, Upstream of Canonical Initiation at AUG*

    PubMed Central

    Fecher-Trost, Claudia; Wissenbach, Ulrich; Beck, Andreas; Schalkowsky, Pascal; Stoerger, Christof; Doerr, Janka; Dembek, Anna; Simon-Thomas, Martin; Weber, Armin; Wollenberg, Peter; Ruppert, Thomas; Middendorff, Ralf; Maurer, Hans H.; Flockerzi, Veit

    2013-01-01

    TRPV6 channels function as epithelial Ca2+ entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca2+ entry to prevent deleterious Ca2+ overload. PMID:23612980

  7. Physiological functions of MTA family of proteins.

    PubMed

    Sen, Nirmalya; Gui, Bin; Kumar, Rakesh

    2014-12-01

    Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

  8. Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

    PubMed Central

    Hung, Chuan-Tien; Kung, Yu-An; Li, Mei-Ling; Lee, Kuo-Ming; Liu, Shih-Tung; Shih, Shin-Ru

    2016-01-01

    The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection. PMID:27780225

  9. BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes Along Different Microtubule Tracks

    PubMed Central

    Guardia, Carlos M.; Farías, Ginny G.; Jia, Rui; Pu, Jing; Bonifacino, Juan S.

    2016-01-01

    Summary The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multisubunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. PMID:27851960

  10. Profiling of conserved non-coding elements upstream of SHOX and functional characterisation of the SHOX cis-regulatory landscape

    PubMed Central

    Verdin, Hannah; Fernández-Miñán, Ana; Benito-Sanz, Sara; Janssens, Sandra; Callewaert, Bert; Waele, Kathleen De; Schepper, Jean De; François, Inge; Menten, Björn; Heath, Karen E.; Gómez-Skarmeta, José Luis; Baere, Elfride De

    2015-01-01

    Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox (SHOX) gene, regulated by seven CNEs located downstream and upstream of SHOX, with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis-regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS, and (iii) the chromatin architecture of the SHOX cis-regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells, and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis-regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis-regulatory domain spanning more than 1 Mb and harbouring putative new cis-regulatory elements. PMID:26631348

  11. Upregulation of Far Upstream Element-Binding Protein 1 (FUBP1) Promotes Tumor Proliferation and Tumorigenesis of Clear Cell Renal Cell Carcinoma

    PubMed Central

    Duan, Junyao; Bao, Xu; Ma, Xin; Zhang, Yu; Ni, Dong; Wang, Hanfeng; Zhang, Fan; Du, Qingshan; Fan, Yang; Chen, Jianwen; Wu, Shengpan; Li, Xintao; Gao, Yu

    2017-01-01

    Objective The far upstream element (FUSE)-binding protein 1 (FUBP1) is a transactivator of human c-myc proto-oncogene transcription, with important roles in carcinogenesis. However, the expression pattern and potential biological function of FUBP1 in clear cell renal cell carcinoma (ccRCC) is yet to be established. Methods FUBP1 expression was detected in ccRCC tissues and cell lines by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The correlations of FUBP1 mRNA expression levels with clinicopathological factors were evaluated. The biological function of FUBP1 during tumor cell proliferation was studied by MTS, colony formation, and soft-agar colony formation. The effects of FUBP1 on cell cycle distribution and apoptosis were analyzed by flow cytometry. Western blot analysis was used to identify the potential mechanism of FUBP1 regulating cell cycle and apoptosis. Results The levels of FUBP1 mRNA and protein expression were upregulated in human ccRCC tissues compared with adjacent noncancerous tissues. High levels of FUBP1 mRNA expression were associated with higher tumor stage and tumor size. FUBP1 knockdown inhibited cell proliferation and induced cell cycle arrest and apoptosis. Meanwhile, the expression levels of c-myc and p21 mRNA were correlated with that of FUBP1 mRNA. Conclusions FUBP1 acts as a potential oncogene in ccRCC and may be considered as a novel biomarker or an attractive treatment target of ccRCC. PMID:28076379

  12. Diverse amino acid residues function within the type 1 peroxisomal targeting signal. Implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal.

    PubMed

    Mullen, R T; Lee, M S; Flynn, C R; Trelease, R N

    1997-11-01

    The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/ L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.

  13. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements.

    PubMed Central

    Liu, B; Stinski, M F

    1992-01-01

    The tegument proteins of human cytomegalovirus are introduced into cells as components of infectious virus. The tegument proteins may affect viral and cellular transcription prior to the synthesis of the immediate-early viral regulatory proteins. The phosphorylated tegument protein of 71 kDa (pp71) is reported to be encoded by the UL82 gene. The UL82 gene products transactivated promoters containing upstream ATF or AP-1 binding sites. In contrast, the phosphorylated tegument protein of 65 kDa (pp65), encoded by the UL83 gene, had no detectable effect on these promoters. Enhancement by UL82 of downstream transcription was directly proportional to the number of upstream ATF sites. Response to UL82 transactivation was abolished by mutation of the ATF site. Mutation in the carboxy-terminal region of UL82 also eliminated transactivation. Even though the major immediate-early promoter of human cytomegalovirus is a strong enhancer-containing promoter, UL82 further enhanced its transcription as much as 20-fold. The mechanism of UL82 enhancement of transcription from viral or cellular promoters is not known, but the enhancement may be mediated by triggering one of the protein kinase signaling pathways, increasing the affinity of ATF or AP-1 for the target sequence, or stabilizing the complex between the eucaryotic transcription factor and the target sequence. Images PMID:1318413

  14. [Protein phosphatases: structure and function].

    PubMed

    Bulanova, E G; Budagian, V M

    1994-01-01

    The process of protein and enzyme systems phosphorylation is necessary for cell growth, differentiation and preparation for division and mitosis. The conformation changes of protein as a result of phosphorylation lead to increased enzyme activity and enhanced affinity to substrates. A large group of enzymes--protein kinases--is responsible for phosphorylation process in cell, which are divided into tyrosine- and serine-threonine-kinases depending on their ability to phosphorylate appropriate amino acid residues. In this review has been considered the functional importance and structure of protein phosphatases--enzymes, which are functional antagonists of protein kinases.

  15. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  16. Functional domains in tetraspanin proteins.

    PubMed

    Stipp, Christopher S; Kolesnikova, Tatiana V; Hemler, Martin E

    2003-02-01

    Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

  17. A Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) Complex Represses Expression of the Gene Encoding Tumor Necrosis Factor α-induced Protein 8 (TNFAIP8)*♦S⃞

    PubMed Central

    Zhang, Ling-juan; Liu, Xiao; Gafken, Philip R.; Kioussi, Chrissa; Leid, Mark

    2009-01-01

    The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) plays key roles in development and homeostasis. A tandem affinity purification procedure revealed that COUP-TFI associated with a number of transcriptional regulatory proteins in HeLa S3 cells, including the nuclear receptor corepressor (NCoR), TIF1β/KAP-1, HDAC1, and the SWI/SNF family member Brahma. The proapoptotic protein DBC1 was also identified in COUP-TFI complexes. In vitro experiments revealed that COUP-TFI interacted directly with NCoR but in a manner different from that of other nuclear receptors. DBC1 stabilized the interaction between COUP-TFI and NCoR by interacting directly with both proteins. The gene encoding the anti-apoptotic protein TNFAIP8 (tumor necrosis factor α (TNFα)-induced protein 8) was identified as being repressed by COUP-TFI in a manner that required several of the component proteins of the COUP-TFI complex. Finally, our studies highlight a central role for COUP-TFI in the induction of the TNFAIP8 promoter by TNFα. Together, these studies identify a novel COUP-TFI complex that functions as a repressor of transcription and may play a role in the TNFα signaling pathways. PMID:19112178

  18. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-01

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  19. Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

    PubMed

    Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

    2015-02-01

    The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.

  20. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules.

    PubMed

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  1. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    SciTech Connect

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  2. Protein kinase C is involved with upstream signaling of methyl farnesoate for photoperiod-dependent sex determination in the water flea Daphnia pulex.

    PubMed

    Toyota, Kenji; Sato, Tomomi; Tatarazako, Norihisa; Iguchi, Taisen

    2017-02-15

    Sex determination of Daphnia pulex is decided by environmental conditions. We established a suitable experimental system for this study using D. pulex WTN6 strain, in which the sex of the offspring can be controlled by photoperiod. Long-day conditions induced females and short-day conditions induced males. Using this system, we previously found that methy farnesoate (MF), which is a putative innate juvenile hormone molecule in daphnids, is necessary for male sex determination and that protein kinase C (PKC) is a candidate factor of male sex determiner. In this study, we demonstrated that a PKC inhibitor [bisindolylmaleimide IV (BIM)] application strongly suppressed male offspring induction in the short-day condition. Moreover, co-treatment of BIM with MF revealed that PKC signaling acts upstream of MF signaling for male sex determination. This is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species.

  3. Protein kinase C is involved with upstream signaling of methyl farnesoate for photoperiod-dependent sex determination in the water flea Daphnia pulex

    PubMed Central

    Toyota, Kenji; Sato, Tomomi; Tatarazako, Norihisa

    2017-01-01

    ABSTRACT Sex determination of Daphnia pulex is decided by environmental conditions. We established a suitable experimental system for this study using D. pulex WTN6 strain, in which the sex of the offspring can be controlled by photoperiod. Long-day conditions induced females and short-day conditions induced males. Using this system, we previously found that methy farnesoate (MF), which is a putative innate juvenile hormone molecule in daphnids, is necessary for male sex determination and that protein kinase C (PKC) is a candidate factor of male sex determiner. In this study, we demonstrated that a PKC inhibitor [bisindolylmaleimide IV (BIM)] application strongly suppressed male offspring induction in the short-day condition. Moreover, co-treatment of BIM with MF revealed that PKC signaling acts upstream of MF signaling for male sex determination. This is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species. PMID:27965197

  4. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases (MKKs) and mitogen activated kinase phosphatase-1 (MKP-1) in microglial cells

    PubMed Central

    Crittenden, Patrick L.; Filipov, Nikolay M.

    2010-01-01

    Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK, and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e., MKK-3/6, MKK-1/2, and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250μM), LPS (100 ng/ml), or Mn+LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn+LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn+LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn+LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4’s phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn+LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn+LPS markedly increased TNF-α , IL-6, and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6, and MKK-4 are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs farthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect. PMID:20589745

  5. Design of membrane proteins: toward functional systems.

    PubMed

    Ghirlanda, Giovanna

    2009-12-01

    Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.

  6. Identification of a novel distal control region upstream of the human steroidogenic acute regulatory protein (StAR) gene that participates in SF-1-dependent chromatin architecture.

    PubMed

    Mizutani, Tetsuya; Yazawa, Takashi; Ju, Yunfeng; Imamichi, Yoshitaka; Uesaka, Miki; Inaoka, Yoshihiko; Matsuura, Kaoru; Kamiki, Yasue; Oki, Masaya; Umezawa, Akihiro; Miyamoto, Kaoru

    2010-09-03

    StAR (steroidogenic acute regulatory protein) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of which is the rate-limiting step for steroidogenesis. Transcriptional regulation of the proximal promoter of the human StAR gene has been well characterized, whereas analysis of its distal control region has not. Recently, we found that SF-1 (steroidogenic factor 1) induced the differentiation of mesenchymal stem cells (MSCs) into steroidogenic cells with the concomitant strong induction of StAR expression. Here, we show, using differentiated MSCs, that StAR expression is regulated by a novel distal control region. Using electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, we identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of StAR. A luciferase reporter assay revealed that the region worked as a strong regulator to exert maximal transcription of StAR. ChIP analysis of histone H3 revealed that upon SF-1 expression, nucleosome eviction took place at the SF-1 binding sites, not only in the promoter but also in the distal SF-1 binding sites. Chromosome conformation capture analysis revealed that the region upstream of StAR formed a chromatin loop both in the differentiated MSCs and in KGN cells, a human granulosa cell tumor cell line, where SF-1 is endogenously expressed. Finally, SF-1 knockdown resulted in disrupted formation of this chromatin loop in KGN cells. These results indicate that the novel distal control region participate in StAR activation through SF-1 dependent alterations of chromatin structure, including histone eviction and chromatin loop formation.

  7. An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo.

    PubMed

    Dorokhov, Yu L; Ivanov, P A; Komarova, T V; Skulachev, M V; Atabekov, J G

    2006-09-01

    It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)(CP,148)(CR) upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem-loop (SL) region of CP subgenomic promoters (TMV U1-CP-GFP/SL-mut and crTMV-CP-GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP-GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP-GFP/SL-mut, whereas a small amount of CP-GFP synthesis was obtained in crTMV-CP-GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.

  8. Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of Kluyveromyces lactis: Characterization of Upstream Region-Activating Sequences in the KIPHO5 Gene

    PubMed Central

    Fermiñán, Encarnación; Domínguez, Angel

    1998-01-01

    Transcription of the repressible acid phosphatase gene (KIPHO5) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPHO5 expression and to design new expression-secretion systems in K. lactis. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPHO5 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (tGH-II), the promoter and signal peptide-encoding region of the phosphate-repressible KIPHO5 gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the Pi present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the Pi concentration. PMID:9647807

  9. The far-upstream element-binding protein 2 is correlated with proliferation and doxorubicin resistance in human breast cancer cell lines.

    PubMed

    Wang, Ying-Ying; Gu, Xiao-Ling; Wang, Chao; Wang, Hua; Ni, Qi-Chao; Zhang, Chun-Hui; Yu, Xia-Fei; Yang, Li-Yi; He, Zhi-Xian; Mao, Guo-Xin; Yang, Shu-Yun

    2016-07-01

    Far-upstream element (FUSE)-binding protein 2 (FBP2) was a member of single-stranded DNA-binding protein family; it played an important role in regulating transcription and post-transcription and is involved in the regulation of C-MYC gene expression in liver tumors. However, the role of FBP2 in breast cancer and its mechanism has not been studied yet. Here, we discovered that FBP2 was up-regulated in breast cancer tissues and breast cancer cell lines. Moreover, immunohistochemistry analysis demonstrated that up-regulated FBP2 was highly associated with tumor grade, Ki-67, and poor prognosis, which was an independent prognostic factor for survival of breast cancer patients. At the cellular level, we found that FBP2 was correlated with cell cycle progression by accelerating G1/S transition, and knockdown of FBP2 could weaken cell proliferation, anchorage-independent cell growth, while enhancing the sensitivity of breast cancer cells to doxorubicin. More importantly, we found that activation of PI3K/AKT pathway could phosphorylate FBP2, and then make FBP2 shuttle from cytoplasm into the nucleus, which was the main mechanism of breast cancer cell proliferation and drug resistance. Taken together, our findings supported the notion that FBP2 might via PI3K/AKT pathway influence breast cancer progression and drug resistance, which might provide a new target for the design of anti-cancer drugs for breast cancer patients.

  10. A statistical study into the spatial distribution and dawn-dusk asymmetry of dayside magnetosheath ion temperatures as a function of upstream solar wind conditions

    NASA Astrophysics Data System (ADS)

    Dimmock, A. P.; Nykyri, K.; Karimabadi, H.; Osmane, A.; Pulkkinen, T. I.

    2015-04-01

    The magnetosheath contains the shocked solar wind and behaves as a natural filter to the solar wind plasma before it reaches the magnetosphere. The redistribution of kinetic energy at the bow shock results in significant thermalization of the solar wind plasma, resulting in a magnetosheath temperature profile which is highly nonhomogeneous and nonisotropic and differs between the dawn and dusk flanks. The present study attempts to study the spatial distribution of magnetosheath ion temperature as a function of upstream solar wind conditions. We pay particular attention to the dawn/dusk asymmetry in which we attempt to quantify using experimental data collected over a 7 year period. We also compare these data to simulated data from both the Block-Adaptive-Tree-Solarwind-Roe-Upwind-Scheme (BATS-R-US) MHD code and a kinetic hybrid model. We present evidence that the dawn flank is consistently hotter than the dusk flank for a variety of upstream conditions. Our statistical data also suggest a dependency on solar wind speed such that the level of asymmetry increases with faster speeds. We conclude that the dawn-favored asymmetry of the magnetosheath seed population is insufficient to explain the dawn asymmetry (30-40%) of cold component ions in the cold, dense plasma sheet, and therefore, other mechanisms are likely required.

  11. Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants.

    PubMed

    Szabados, L; Ratet, P; Grunenberg, B; de Bruijn, F J

    1990-10-01

    Expression of the Sesbania rostrata leghemoglobin glb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA (gus) gene fusions to identify cis-acting elements involved in nodule-specific gene expression and general transcriptional control. A 1.9-kilobase fragment of the glb3 5'-upstream region was found to direct a high level of nodule-specific beta-glucuronidase (GUS) activity in L. corniculatus, restricted to the Rhizobium-infected cells of the nodules. The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system. A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression. Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters delta-p35S and delta-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L. corniculatus but a significant increase in tobacco, primarily in the roots. The same fragment could not direct nodule-specific expression when fused to a heterologous enhancer in cis. This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L. corniculatus that function poorly or may be involved in promoter silencing in tobacco. By fusing further upstream fragments to the delta-p35S and delta-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well as -429 and -162. The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L. corniculatus and tobacco. The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L. corniculatus. These results show that efficient expression of the S. rostrata glb3 gene in nodules is

  12. Phospholipid liposomes functionalized by protein

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Savostyanov, G. V.; Grishina, O. A.

    2015-03-01

    Finding new ways to deliver neurotrophic drugs to the brain in newborns is one of the contemporary problems of medicine and pharmaceutical industry. Modern researches in this field indicate the promising prospects of supramolecular transport systems for targeted drug delivery to the brain which can overcome the blood-brain barrier (BBB). Thus, the solution of this problem is actual not only for medicine, but also for society as a whole because it determines the health of future generations. Phospholipid liposomes due to combination of lipo- and hydrophilic properties are considered as the main future objects in medicine for drug delivery through the BBB as well as increasing their bioavailability and toxicity. Liposomes functionalized by various proteins were used as transport systems for ease of liposomes use. Designing of modification oligosaccharide of liposomes surface is promising in the last decade because it enables the delivery of liposomes to specific receptor of human cells by selecting ligand and it is widely used in pharmacology for the treatment of several diseases. The purpose of this work is creation of a coarse-grained model of bilayer of phospholipid liposomes, functionalized by specific to the structural elements of the BBB proteins, as well as prediction of the most favorable orientation and position of the molecules in the generated complex by methods of molecular docking for the formation of the structure. Investigation of activity of the ligand molecule to protein receptor of human cells by the methods of molecular dynamics was carried out.

  13. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    PubMed Central

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  14. Functional Whole-genome Analysis Identifies Polo-like Kinase 2 and Poliovirus Receptor as Essential for Neuronal Differentiation Upstream of the Negative Regulator αB-crystallin

    PubMed Central

    Draghetti, Cristina; Salvat, Catherine; Zanoguera, Francisca; Curchod, Marie-Laure; Vignaud, Chloé; Peixoto, Helene; Di Cara, Alessandro; Fischer, David; Dhanabal, Mohanraj; Andreas, Goutopoulos; Abderrahim, Hadi; Rommel, Christian; Camps, Montserrat

    2009-01-01

    This study aimed at identifying transcriptional changes associated to neuronal differentiation induced by six distinct stimuli using whole-genome microarray hybridization analysis. Bioinformatics analyses revealed the clustering of these six stimuli into two categories, suggesting separate gene/pathway dependence. Treatment with specific inhibitors demonstrated the requirement of both Janus kinase and microtubule-associated protein kinase activation to trigger differentiation with nerve growth factor (NGF) and dibutyryl cAMP. Conversely, activation of protein kinase A, phosphatidylinositol-3-kinase α, and mammalian target of rapamycin, although required for dibutyryl cAMP-induced differentiation, exerted a negative feedback on NGF-induced differentiation. We identified Polo-like kinase 2 (Plk2) and poliovirus receptor (PVR) as indispensable for NGF-driven neuronal differentiation and αB-crystallin (Cryab) as an inhibitor of this process. Silencing of Plk2 or PVR blocked NGF-triggered differentiation and Cryab down-regulation, while silencing of Cryab enhanced NGF-induced differentiation. Our results position both Plk2 and PVR upstream of the negative regulator Cryab in the pathway(s) leading to neuronal differentiation triggered by NGF. PMID:19700763

  15. Metastasis-associated protein 1 is an upstream regulator of DNMT3a and stimulator of insulin-growth factor binding protein-3 in breast cancer.

    PubMed

    Deivendran, S; Marzook, Hezlin; Santhoshkumar, T R; Kumar, Rakesh; Pillai, M Radhakrishna

    2017-04-10

    Despite a recognized role of DNA methyltransferase 3a (DNMT3a) in human cancer, the nature of its upstream regulator(s) and relationship with the master chromatin remodeling factor MTA1, continues to be poorly understood. Here, we found an inverse relationship between the levels of MTA1 and DNMT3a in human cancer and that high levels of MTA1 in combination of low DNMT3a status correlates well with poor survival of breast cancer patients. We discovered that MTA1 represses DNMT3a expression via HDAC1/YY1 transcription factor complex. Because IGFBP3 is an established target of DNMT3a, we investigated the effect of MTA1 upon IGFBP3 expression, and found a coactivator role of MTA1/c-Jun/Pol II coactivator complex upon the IGFBP3 transcription. In addition, MTA1 overexpression correlates well with low levels of DNMT3a which, in turn also correlates with a high IGFBP3 status in breast cancer patients and predicts a poor clinical outcome for breast cancer patients. These findings suggest that MTA1 could regulate the expression of IGFBP3 in both DNMT3a-dependent and -independent manner. Together findings presented here recognize an inherent role of MTA1 as a modifier of DNMT3a and IGFBP3 expression, and consequently, the role of MTA1-DNMT3a-IGFBP3 axis in breast cancer progression.

  16. Metastasis-associated protein 1 is an upstream regulator of DNMT3a and stimulator of insulin-growth factor binding protein-3 in breast cancer

    PubMed Central

    Deivendran, S.; Marzook, Hezlin; Santhoshkumar, T. R.; Kumar, Rakesh; Pillai, M. Radhakrishna

    2017-01-01

    Despite a recognized role of DNA methyltransferase 3a (DNMT3a) in human cancer, the nature of its upstream regulator(s) and relationship with the master chromatin remodeling factor MTA1, continues to be poorly understood. Here, we found an inverse relationship between the levels of MTA1 and DNMT3a in human cancer and that high levels of MTA1 in combination of low DNMT3a status correlates well with poor survival of breast cancer patients. We discovered that MTA1 represses DNMT3a expression via HDAC1/YY1 transcription factor complex. Because IGFBP3 is an established target of DNMT3a, we investigated the effect of MTA1 upon IGFBP3 expression, and found a coactivator role of MTA1/c-Jun/Pol II coactivator complex upon the IGFBP3 transcription. In addition, MTA1 overexpression correlates well with low levels of DNMT3a which, in turn also correlates with a high IGFBP3 status in breast cancer patients and predicts a poor clinical outcome for breast cancer patients. These findings suggest that MTA1 could regulate the expression of IGFBP3 in both DNMT3a-dependent and -independent manner. Together findings presented here recognize an inherent role of MTA1 as a modifier of DNMT3a and IGFBP3 expression, and consequently, the role of MTA1-DNMT3a-IGFBP3 axis in breast cancer progression. PMID:28393842

  17. An iterative approach of protein function prediction

    PubMed Central

    2011-01-01

    Background Current approaches of predicting protein functions from a protein-protein interaction (PPI) dataset are based on an assumption that the available functions of the proteins (a.k.a. annotated proteins) will determine the functions of the proteins whose functions are unknown yet at the moment (a.k.a. un-annotated proteins). Therefore, the protein function prediction is a mono-directed and one-off procedure, i.e. from annotated proteins to un-annotated proteins. However, the interactions between proteins are mutual rather than static and mono-directed, although functions of some proteins are unknown for some reasons at present. That means when we use the similarity-based approach to predict functions of un-annotated proteins, the un-annotated proteins, once their functions are predicted, will affect the similarities between proteins, which in turn will affect the prediction results. In other words, the function prediction is a dynamic and mutual procedure. This dynamic feature of protein interactions, however, was not considered in the existing prediction algorithms. Results In this paper, we propose a new prediction approach that predicts protein functions iteratively. This iterative approach incorporates the dynamic and mutual features of PPI interactions, as well as the local and global semantic influence of protein functions, into the prediction. To guarantee predicting functions iteratively, we propose a new protein similarity from protein functions. We adapt new evaluation metrics to evaluate the prediction quality of our algorithm and other similar algorithms. Experiments on real PPI datasets were conducted to evaluate the effectiveness of the proposed approach in predicting unknown protein functions. Conclusions The iterative approach is more likely to reflect the real biological nature between proteins when predicting functions. A proper definition of protein similarity from protein functions is the key to predicting functions iteratively. The

  18. Far upstream element-binding protein 1 (FUBP1) is a potential c-Myc regulator in esophageal squamous cell carcinoma (ESCC) and its expression promotes ESCC progression.

    PubMed

    Yang, Lei; Zhu, Jun-Ya; Zhang, Jian-Guo; Bao, Bo-Jun; Guan, Cheng-Qi; Yang, Xiao-Jing; Liu, Yan-Hua; Huang, Yue-Jiao; Ni, Run-Zhou; Ji, Li-Li

    2016-03-01

    The human far upstream element (FUSE) binding protein 1 (FUBP1) belongs to an ancient family which is required for proper regulation of the c-Myc proto-oncogene. Although c-Myc plays an important role in development of various carcinomas, the relevance of FUBP1 and their contribution to esophageal squamous cell carcinoma (ESCC) development remain unclear. In this study, we aimed to investigate the relationship between FUBP1 and c-Myc as well as their contribution to ESCC development. Western blot and immunohistochemical analyses were performed to evaluate FUBP1 expression. Coimmunoprecipitation analysis was performed to explore the correlation between FUBP1 and c-Myc in ESCC. In addition, the role of FUBP1 in ESCC proliferation was studied in ESCC cells through knocking FUBP1 down. The regulation of FUBP1 on proliferation was confirmed by Cell Counting Kit-8 (CCK-8) assay, flow cytometric assays, and clone formation assays. The expressions of FUBP1 and c-Myc were both upregulated in ESCC tissues. In addition to correlation between expression of FUBP1 and tumor grade, we also confirmed the correlation of FUBP1, c-Myc, and Ki-67 expression by twos. Moreover, upregulation of FUBP1 and c-Myc in ESCC was associated with poor survival. FUBP1 was confirmed to activate c-Myc in ESCC tissues and cells. FUBP1 was demonstrated to promote proliferation of ESCC cells. Moreover, downregulation of both FUBP1 and c-Myc was confirmed to inhibit proliferation of ESCC cells. Our results indicated that FUBP1 may potentially stimulate c-Myc expression in ESCC and its expression may promote ESCC progression.

  19. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

  20. Functional assignment to JEV proteins using SVM

    PubMed Central

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

  1. Functional Foods Containing Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whey proteins, modified whey proteins, and whey components are useful as nutrients or supplements for health maintenance. Extrusion modified whey proteins can easily fit into new products such as beverages, confectionery items (e.g., candies), convenience foods, desserts, baked goods, sauces, and in...

  2. Upstream health law.

    PubMed

    Sage, William M; McIlhattan, Kelley

    2014-01-01

    For the first time, entrepreneurs are aggressively developing new technologies and business models designed to improve individual and population health, not just to deliver specialized medical care. Consumers of these goods and services are not yet "patients"; they are simply people. As this sector of the health care industry expands, it is likely to require new forms of legal governance, which we term "upstream health law."

  3. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  4. Prediction of protein function from protein sequence and structure.

    PubMed

    Whisstock, James C; Lesk, Arthur M

    2003-08-01

    The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as

  5. Protein Function Prediction: Problems and Pitfalls.

    PubMed

    Pearson, William R

    2015-09-03

    The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified proteins. Here, we highlight some of the challenges in functional inference from sequence similarity. Investigators can improve the accuracy of function prediction by (1) being conservative about the evolutionary distance to a protein of known function; (2) considering the ambiguous meaning of "functional similarity," and (3) being aware of the limitations of annotations in functional databases. Protein function prediction does not offer "one-size-fits-all" solutions. Prediction strategies work better when the idiosyncrasies of function and functional annotation are better understood.

  6. Exploring Mouse Protein Function via Multiple Approaches

    PubMed Central

    Huang, Tao; Kong, Xiangyin; Zhang, Yunhua; Zhang, Ning

    2016-01-01

    Although the number of available protein sequences is growing exponentially, functional protein annotations lag far behind. Therefore, accurate identification of protein functions remains one of the major challenges in molecular biology. In this study, we presented a novel approach to predict mouse protein functions. The approach was a sequential combination of a similarity-based approach, an interaction-based approach and a pseudo amino acid composition-based approach. The method achieved an accuracy of about 0.8450 for the 1st-order predictions in the leave-one-out and ten-fold cross-validations. For the results yielded by the leave-one-out cross-validation, although the similarity-based approach alone achieved an accuracy of 0.8756, it was unable to predict the functions of proteins with no homologues. Comparatively, the pseudo amino acid composition-based approach alone reached an accuracy of 0.6786. Although the accuracy was lower than that of the previous approach, it could predict the functions of almost all proteins, even proteins with no homologues. Therefore, the combined method balanced the advantages and disadvantages of both approaches to achieve efficient performance. Furthermore, the results yielded by the ten-fold cross-validation indicate that the combined method is still effective and stable when there are no close homologs are available. However, the accuracy of the predicted functions can only be determined according to known protein functions based on current knowledge. Many protein functions remain unknown. By exploring the functions of proteins for which the 1st-order predicted functions are wrong but the 2nd-order predicted functions are correct, the 1st-order wrongly predicted functions were shown to be closely associated with the genes encoding the proteins. The so-called wrongly predicted functions could also potentially be correct upon future experimental verification. Therefore, the accuracy of the presented method may be much higher in

  7. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  8. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  9. Origins of Protein Functions in Cells

    NASA Technical Reports Server (NTRS)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

  10. CEH-20/Pbx and UNC-62/Meis function upstream of rnt-1/Runx to regulate asymmetric divisions of the C. elegans stem-like seam cells.

    PubMed

    Hughes, Samantha; Brabin, Charles; Appleford, Peter J; Woollard, Alison

    2013-07-15

    Caenorhabditis elegans seam cells divide in the stem-like mode throughout larval development, with the ability to both self-renew and produce daughters that differentiate. Seam cells typically divide asymmetrically, giving rise to an anterior daughter that fuses with the hypodermis and a posterior daughter that proliferates further. Previously we have identified rnt-1 (a homologue of the mammalian cancer-associated stem cell regulator Runx) as being an important regulator of seam development, acting to promote proliferation; rnt-1 mutants have fewer seam cells whereas overexpressing rnt-1 causes seam cell hyperplasia. We isolated the interacting CEH-20/Pbx and UNC-62/Meis TALE-class transcription factors during a genome-wide RNAi screen for novel regulators of seam cell number. Animals lacking wild type CEH-20 or UNC-62 display seam cell hyperplasia, largely restricted to the anterior of the worm, whereas double mutants have many additional seam cells along the length of the animal. The cellular basis of the hyperplasia involves the symmetrisation of normally asymmetric seam cell divisions towards the proliferative stem-like fate. The hyperplasia is completely suppressed in rnt-1 mutants, and rnt-1 is upregulated in ceh-20 and unc-62 mutants, suggesting that CEH-20 and UNC-62 function upstream of rnt-1 to limit proliferative potential to the appropriate daughter cell. In further support of this we find that CEH-20 is asymmetrically localised in seam daughters following an asymmetric division, being predominantly restricted to anterior nuclei whose fate is to differentiate. Thus, ceh-20 and unc-62 encode crucial regulators of seam cell division asymmetry, acting via rnt-1 to regulate the balance between proliferation and differentiation.

  11. Year 2 Report: Protein Function Prediction Platform

    SciTech Connect

    Zhou, C E

    2012-04-27

    Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fully automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.

  12. Detection of Functional Modes in Protein Dynamics

    PubMed Central

    Hub, Jochen S.; de Groot, Bert L.

    2009-01-01

    Proteins frequently accomplish their biological function by collective atomic motions. Yet the identification of collective motions related to a specific protein function from, e.g., a molecular dynamics trajectory is often non-trivial. Here, we propose a novel technique termed “functional mode analysis” that aims to detect the collective motion that is directly related to a particular protein function. Based on an ensemble of structures, together with an arbitrary “functional quantity” that quantifies the functional state of the protein, the technique detects the collective motion that is maximally correlated to the functional quantity. The functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. In addition, the motion that displays the largest likelihood to induce a substantial change in the functional quantity is estimated from the given protein ensemble. Two different correlation measures are applied: first, the Pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. Detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. The new approach is illustrated using a number of biomolecules, including a polyalanine-helix, T4 lysozyme, Trp-cage, and leucine-binding protein. PMID:19714202

  13. Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts.

    PubMed

    Fulton, Daniel C; Stettler, Michaela; Mettler, Tabea; Vaughan, Cara K; Li, Jing; Francisco, Perigio; Gil, Manuel; Reinhold, Heike; Eicke, Simona; Messerli, Gaëlle; Dorken, Gary; Halliday, Karen; Smith, Alison M; Smith, Steven M; Zeeman, Samuel C

    2008-04-01

    This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

  14. Characterization and Functionality of Corn Germ Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to evaluate the functional properties of protein extracted from wet-milled corn germ and identify potential applications of the recovered protein. Corn germ comprises 12% of the total weight of normal dent corn and about 29% of the corn protein (moisture-free and oil- free ...

  15. Protein function prediction based on data fusion and functional interrelationship.

    PubMed

    Meng, Jun; Wekesa, Jael-Sanyanda; Shi, Guan-Li; Luan, Yu-Shi

    2016-04-01

    One of the challenging tasks of bioinformatics is to predict more accurate and confident protein functions from genomics and proteomics datasets. Computational approaches use a variety of high throughput experimental data, such as protein-protein interaction (PPI), protein sequences and phylogenetic profiles, to predict protein functions. This paper presents a method that uses transductive multi-label learning algorithm by integrating multiple data sources for classification. Multiple proteomics datasets are integrated to make inferences about functions of unknown proteins and use a directed bi-relational graph to assign labels to unannotated proteins. Our method, bi-relational graph based transductive multi-label function annotation (Bi-TMF) uses functional correlation and topological PPI network properties on both the training and testing datasets to predict protein functions through data fusion of the individual kernel result. The main purpose of our proposed method is to enhance the performance of classifier integration for protein function prediction algorithms. Experimental results demonstrate the effectiveness and efficiency of Bi-TMF on multi-sources datasets in yeast, human and mouse benchmarks. Bi-TMF outperforms other recently proposed methods.

  16. J domain independent functions of J proteins.

    PubMed

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

    Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

  17. Functions of red cell surface proteins.

    PubMed

    Daniels, G

    2007-11-01

    The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.

  18. Repressors and Upstream Repressing Sequences of the Stress-Regulated ENA1 Gene in Saccharomyces cerevisiae: bZIP Protein Sko1p Confers HOG-Dependent Osmotic Regulation

    PubMed Central

    Proft, Markus; Serrano, Ramón

    1999-01-01

    The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (−533 to −544) which was characterized as an upstream repressing sequence (URSMIG-ENA1) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at −502 to −513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URSCRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase–Sko1 fusion protein binds specifically to the URSCRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URSCRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URSCRE-ENA1 and URSMIG-ENA1) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or

  19. Food Protein Functionality--A New Model.

    PubMed

    Foegeding, E Allen

    2015-12-01

    Proteins in foods serve dual roles as nutrients and structural building blocks. The concept of protein functionality has historically been restricted to nonnutritive functions--such as creating emulsions, foams, and gels--but this places sole emphasis on food quality considerations and potentially overlooks modifications that may also alter nutritional quality or allergenicity. A new model is proposed that addresses the function of proteins in foods based on the length scale(s) responsible for the function. Properties such as flavor binding, color, allergenicity, and digestibility are explained based on the structure of individual molecules; placing this functionality at the nano/molecular scale. At the next higher scale, applications in foods involving gelation, emulsification, and foam formation are based on how proteins form secondary structures that are seen at the nano and microlength scales, collectively called the mesoscale. The macroscale structure represents the arrangements of molecules and mesoscale structures in a food. Macroscale properties determine overall product appearance, stability, and texture. The historical approach of comparing among proteins based on forming and stabilizing specific mesoscale structures remains valid but emphasis should be on a common means for structure formation to allow for comparisons across investigations. For applications in food products, protein functionality should start with identification of functional needs across scales. Those needs are then evaluated relative to how processing and other ingredients could alter desired molecular scale properties, or proper formation of mesoscale structures. This allows for a comprehensive approach to achieving the desired function of proteins in foods.

  20. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  1. Damping and spectral formation of upstream whistlers

    SciTech Connect

    Orlowski, D.S.; Russell, C.T.; Krauss-Varban, D.

    1995-09-01

    Previous studies have indicated that damping rates of upstream whistlers strongly depend on the details of the electron distribution function. Moreover, detailed analysis of Doppler shift and the whistler dispersion relation indicate that upstream whistlers propagate obliquely in a finite band of frequencies. In this paper we present results of a kinetic calculation of damping lengths of wideband whistlers using the sum of seven drifting bi-Maxwellian electron distributions as a best fit to the ISEE 1 electron data. For two cases, when upstream whistlers are observed, convective damping lengths derived from ISEE magnetic field and ephemeris data are compared with theoretical results. We find that the calculated convective damping lengths are consistent with the data and that upstream whistlers remain marginally stable. We also show that the slope of plasma frame spectra of upstream whistlers, obtained by direct fitting of the observed spectra, is between 5 and 7. The overall spectral, wave, and particle characteristics, proximity to the shock, as well as propagation and damping properties indicated that these waves cannot be generated locally. Instead, the observed upstream whistlers arise in the shock ramp, most likely by a variety of cross-field drift and/or anisotropy driven instabilities. 57 refs., 11 figs.

  2. Phylointeractomics reconstructs functional evolution of protein binding

    PubMed Central

    Kappei, Dennis; Scheibe, Marion; Paszkowski-Rogacz, Maciej; Bluhm, Alina; Gossmann, Toni Ingolf; Dietz, Sabrina; Dejung, Mario; Herlyn, Holger; Buchholz, Frank; Mann, Matthias; Butter, Falk

    2017-01-01

    Molecular phylogenomics investigates evolutionary relationships based on genomic data. However, despite genomic sequence conservation, changes in protein interactions can occur relatively rapidly and may cause strong functional diversification. To investigate such functional evolution, we here combine phylogenomics with interaction proteomics. We develop this concept by investigating the molecular evolution of the shelterin complex, which protects telomeres, across 16 vertebrate species from zebrafish to humans covering 450 million years of evolution. Our phylointeractomics screen discovers previously unknown telomere-associated proteins and reveals how homologous proteins undergo functional evolution. For instance, we show that TERF1 evolved as a telomere-binding protein in the common stem lineage of marsupial and placental mammals. Phylointeractomics is a versatile and scalable approach to investigate evolutionary changes in protein function and thus can provide experimental evidence for phylogenomic relationships. PMID:28176777

  3. Protein function from its emergence to diversity in contemporary proteins

    NASA Astrophysics Data System (ADS)

    Goncearenco, Alexander; Berezovsky, Igor N.

    2015-07-01

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions—elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  4. Protein function from its emergence to diversity in contemporary proteins.

    PubMed

    Goncearenco, Alexander; Berezovsky, Igor N

    2015-06-09

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions-elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  5. Predicting protein function by frequent functional association pattern mining in protein interaction networks.

    PubMed

    Cho, Young-Rae; Zhang, Aidong

    2010-01-01

    Predicting protein function from protein interaction networks has been challenging because of the complexity of functional relationships among proteins. Most previous function prediction methods depend on the neighborhood of or the connected paths to known proteins. However, their accuracy has been limited due to the functional inconsistency of interacting proteins. In this paper, we propose a novel approach for function prediction by identifying frequent patterns of functional associations in a protein interaction network. A set of functions that a protein performs is assigned into the corresponding node as a label. A functional association pattern is then represented as a labeled subgraph. Our frequent labeled subgraph mining algorithm efficiently searches the functional association patterns that occur frequently in the network. It iteratively increases the size of frequent patterns by one node at a time by selective joining, and simplifies the network by a priori pruning. Using the yeast protein interaction network, our algorithm found more than 1400 frequent functional association patterns. The function prediction is performed by matching the subgraph, including the unknown protein, with the frequent patterns analogous to it. By leave-one-out cross validation, we show that our approach has better performance than previous link-based methods in terms of prediction accuracy. The frequent functional association patterns generated in this study might become the foundations of advanced analysis for functional behaviors of proteins in a system level.

  6. Hypoxia-inducible factor 1/heme oxygenase 1 cascade as upstream signals in the prolife role of heat shock protein 70 at rostral ventrolateral medulla during experimental brain stem death.

    PubMed

    Chang, Alice Y W; Chan, Julie Y H; Cheng, Hsiao-Lei; Tsai, Ching-Yi; Chan, Samuel H H

    2009-12-01

    As the origin of a life-and-death signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to delineate the cellular mechanisms of this fateful phenomenon. Based on a clinically relevant animal model that used the organophosphate pesticide mevinphos (Mev) as the experimental insult, we reported previously that heat shock protein 70 (HSP70) in RVLM plays a prolife role by ameliorating circulatory depression during brain stem death. Because Mev also elicits significant hypoxia in RVLM, this study evaluated the hypothesis that the hypoxia-inducible factor 1 (HIF-1)/heme oxygenase 1 (HO-1) cascade acts as upstream signals in the prolife role of HSP70 at RVLM during experimental brain stem death. In Sprague-Dawley rats maintained under propofol anesthesia, transcription activity assay or Western blot analysis revealed an enhancement of nuclear activity of HIF-1alpha or augmentation of HO-1 and HSP70 expression in RVLM preferentially during the prolife phase of Mev intoxication. Loss-of-function manipulations in RVLM using HIF-1alpha, HIF-1beta, or HO-1 antiserum or antisense hif-1alpha or ho-1 oligonucleotide significantly antagonized the preferential upregulation of HSP70, depressed the sustained cardiovascular regulatory machinery during the prolife phase, and exacerbated circulatory depression during the prodeath phase. Immunoneutralization of HIF-1alpha also blunted the preferential increase in HO-1 expression. We conclude that the repertoire of cellular events in RVLM during the prolife phase in our Mev intoxication of brain stem death triggered by hypoxia entails sequential activation of HIF-1, HO-1, and HSP70, leading to neuroprotection by amelioration of cardiovascular depression.

  7. Assigning protein functions by comparative genome analysis protein phylogenetic profiles

    DOEpatents

    Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

    2003-05-13

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  8. Structural determinants of TRIM protein function.

    PubMed

    Esposito, Diego; Koliopoulos, Marios G; Rittinger, Katrin

    2017-02-08

    Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of Really Interesting New Gene (RING) E3 ubiquitin ligases and contribute to the regulation of numerous cellular activities, including innate immune responses. The conserved TRIM harbours a RING domain that imparts E3 ligase activity to TRIM family proteins, whilst a variable C-terminal region can mediate recognition of substrate proteins. The knowledge of the structure of these multidomain proteins and the functional interplay between their constituent domains is paramount to understanding their cellular roles. To date, available structural information on TRIM proteins is still largely restricted to subdomains of many TRIMs in isolation. Nevertheless, applying a combination of structural, biophysical and biochemical approaches has recently allowed important progress to be made towards providing a better understanding of the molecular features that underlie the function of TRIM family proteins and has uncovered an unexpected diversity in the link between self-association and catalytic activity.

  9. A review on protein functionalized carbon nanotubes.

    PubMed

    Nagaraju, Kathyayini; Reddy, Roopa; Reddy, Narendra

    2015-12-18

    Carbon nanotubes (CNTs) have been widely recognized and used for controlled drug delivery and in various other fields due to their unique properties and distinct advantages. Both single-walled carbon nanotubes (SWCNTs) and multiwalled (MWCNTs) carbon nanotubes are used and/or studied for potential applications in medical, energy, textile, composite, and other areas. Since CNTs are chemically inert and are insoluble in water or other organic solvents, they are functionalized or modified to carry payloads or interact with biological molecules. CNTs have been preferably functionalized with proteins because CNTs are predominantly used for medical applications such as delivery of drugs, DNA and genes, and also for biosensing. Extensive studies have been conducted to understand the interactions, cytotoxicity, and potential applications of protein functionalized CNTs but contradicting results have been published on the cytotoxicity of the functionalized CNTs. This paper provides a brief review of CNTs functionalized with proteins, methods used to functionalize the CNTs, and their potential applications.

  10. Using the folding landscapes of proteins to understand protein function.

    PubMed

    Giri Rao, V V Hemanth; Gosavi, Shachi

    2016-02-01

    Proteins fold on a biologically-relevant timescale because of a funnel-shaped energy landscape. This landscape is sculpted through evolution by selecting amino-acid sequences that stabilize native interactions while suppressing stable non-native interactions that occur during folding. However, there is strong evolutionary selection for functional residues and these cannot be chosen to optimize folding. Their presence impacts the folding energy landscape in a variety of ways. Here, we survey the effects of functional residues on folding by providing several examples. We then review how such effects can be detected computationally and be used as assays for protein function. Overall, an understanding of how functional residues modulate folding should provide insights into the design of natural proteins and their homeostasis.

  11. Protein molecular function prediction by Bayesian phylogenomics.

    PubMed

    Engelhardt, Barbara E; Jordan, Michael I; Muratore, Kathryn E; Brenner, Steven E

    2005-10-01

    We present a statistical graphical model to infer specific molecular function for unannotated protein sequences using homology. Based on phylogenomic principles, SIFTER (Statistical Inference of Function Through Evolutionary Relationships) accurately predicts molecular function for members of a protein family given a reconciled phylogeny and available function annotations, even when the data are sparse or noisy. Our method produced specific and consistent molecular function predictions across 100 Pfam families in comparison to the Gene Ontology annotation database, BLAST, GOtcha, and Orthostrapper. We performed a more detailed exploration of functional predictions on the adenosine-5'-monophosphate/adenosine deaminase family and the lactate/malate dehydrogenase family, in the former case comparing the predictions against a gold standard set of published functional characterizations. Given function annotations for 3% of the proteins in the deaminase family, SIFTER achieves 96% accuracy in predicting molecular function for experimentally characterized proteins as reported in the literature. The accuracy of SIFTER on this dataset is a significant improvement over other currently available methods such as BLAST (75%), GeneQuiz (64%), GOtcha (89%), and Orthostrapper (11%). We also experimentally characterized the adenosine deaminase from Plasmodium falciparum, confirming SIFTER's prediction. The results illustrate the predictive power of exploiting a statistical model of function evolution in phylogenomic problems. A software implementation of SIFTER is available from the authors.

  12. Function and structure of inherently disordered proteins.

    PubMed

    Dunker, A Keith; Silman, Israel; Uversky, Vladimir N; Sussman, Joel L

    2008-12-01

    The application of bioinformatics methodologies to proteins inherently lacking 3D structure has brought increased attention to these macromolecules. Here topics concerning these proteins are discussed, including their prediction from amino acid sequence, their enrichment in eukaryotes compared to prokaryotes, their more rapid evolution compared to structured proteins, their organization into specific groups, their structural preferences, their half-lives in cells, their contributions to signaling diversity (via high contents of multiple-partner binding sites, post-translational modifications, and alternative splicing), their distinct functional repertoire compared to that of structured proteins, and their involvement in diseases.

  13. Functional Importance of Mobile Ribosomal Proteins.

    PubMed

    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  14. Control of protein function through optochemical translocation.

    PubMed

    Engelke, Hanna; Chou, Chungjung; Uprety, Rajendra; Jess, Phillip; Deiters, Alexander

    2014-10-17

    Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applications-light-triggered transcription and light-triggered protease cleavage-both based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology.

  15. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    PubMed

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2016-09-12

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

  16. Protein conformational populations and functionally relevant substates.

    PubMed

    Ramanathan, Arvind; Savol, Andrej; Burger, Virginia; Chennubhotla, Chakra S; Agarwal, Pratul K

    2014-01-21

    Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of the protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow

  17. Nano-functionalization of protein microspheres

    NASA Astrophysics Data System (ADS)

    Yoon, Sungkwon; Nichols, William T.

    2014-08-01

    Protein microspheres are promising building blocks for the assembly of complex functional materials. Here we demonstrate a set of three techniques that add functionality to the surface of protein microspheres. In the first technique, a positive surface charge on the protein spheres is deposited by electrostatic adsorption. Negatively charged silica and gold nanoparticle colloids can then electrostatically bind reversibly to the microsphere surface. In the second technique, nanoparticles are covalently anchored to the protein shell using a simple one-pot process. The strong covalent bond between sulfur groups in cysteine in the protein shell irreversibly binds to the gold nanoparticles. In the third technique, surface morphology of the protein microsphere is tuned through hydrodynamic instability at the water-oil interface. This is accomplished through the degree of solubility of the oil phase in water. Taken together these three techniques form a platform to create nano-functionalized protein microspheres, which can then be used as building blocks for the assembly of more complex macroscopic materials.

  18. Evolution-Based Functional Decomposition of Proteins.

    PubMed

    Rivoire, Olivier; Reynolds, Kimberly A; Ranganathan, Rama

    2016-06-01

    The essential biological properties of proteins-folding, biochemical activities, and the capacity to adapt-arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment-a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation.

  19. Upstream of Saturn and Titan

    NASA Astrophysics Data System (ADS)

    Arridge, C. S.; André, N.; Bertucci, C. L.; Garnier, P.; Jackman, C. M.; Németh, Z.; Rymer, A. M.; Sergis, N.; Szego, K.; Coates, A. J.; Crary, F. J.

    The formation of Titan's induced magnetosphere is a unique and important example in the solar system of a plasma-moon interaction where the moon has a substantial atmosphere. The field and particle conditions upstream of Titan are important in controlling the interaction and also play a strong role in modulating the chemistry of the ionosphere. In this paper we review Titan's plasma interaction to identify important upstream parameters and review the physics of Saturn's magnetosphere near Titan's orbit to highlight how these upstream parameters may vary. We discuss the conditions upstream of Saturn in the solar wind and the conditions found in Saturn's magnetosheath. Statistical work on Titan's upstream magnetospheric fields and particles are discussed. Finally, various classification schemes are presented and combined into a single list of Cassini Titan encounter classes which is also used to highlight differences between these classification schemes.

  20. Upstream of Saturn and Titan

    NASA Astrophysics Data System (ADS)

    Arridge, C. S.; André, N.; Bertucci, C. L.; Garnier, P.; Jackman, C. M.; Németh, Z.; Rymer, A. M.; Sergis, N.; Szego, K.; Coates, A. J.; Crary, F. J.

    2011-12-01

    The formation of Titan's induced magnetosphere is a unique and important example in the solar system of a plasma-moon interaction where the moon has a substantial atmosphere. The field and particle conditions upstream of Titan are important in controlling the interaction and also play a strong role in modulating the chemistry of the ionosphere. In this paper we review Titan's plasma interaction to identify important upstream parameters and review the physics of Saturn's magnetosphere near Titan's orbit to highlight how these upstream parameters may vary. We discuss the conditions upstream of Saturn in the solar wind and the conditions found in Saturn's magnetosheath. Statistical work on Titan's upstream magnetospheric fields and particles are discussed. Finally, various classification schemes are presented and combined into a single list of Cassini Titan encounter classes which is also used to highlight differences between these classification schemes.

  1. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  2. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  3. Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2).

    PubMed

    Lackmann, Fredrik; Belikov, Sergey; Wieslander, Lars

    2017-01-01

    Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3.

  4. Investigating neuronal function with optically controllable proteins

    PubMed Central

    Zhou, Xin X.; Pan, Michael; Lin, Michael Z.

    2015-01-01

    In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain. PMID:26257603

  5. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  6. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.

    PubMed

    Fahsold, R; Hoffmeyer, S; Mischung, C; Gille, C; Ehlers, C; Kücükceylan, N; Abdel-Nour, M; Gewies, A; Peters, H; Kaufmann, D; Buske, A; Tinschert, S; Nürnberg, P

    2000-03-01

    More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region

  7. Protein function prediction using neighbor relativity in protein-protein interaction network.

    PubMed

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network.

  8. Protein Nitration in Placenta – Functional Significance

    PubMed Central

    Webster, RP; Roberts, VHJ; Myatt, L

    2009-01-01

    Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings. PMID:18851882

  9. Proteins with Novel Structure, Function and Dynamics

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew

    2014-01-01

    Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

  10. Modular protein domains: an engineering approach toward functional biomaterials.

    PubMed

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

  11. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  12. The lipocalin protein family: structure and function.

    PubMed Central

    Flower, D R

    1996-01-01

    The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds. PMID:8761444

  13. Hierarchical Ensemble Methods for Protein Function Prediction

    PubMed Central

    2014-01-01

    Protein function prediction is a complex multiclass multilabel classification problem, characterized by multiple issues such as the incompleteness of the available annotations, the integration of multiple sources of high dimensional biomolecular data, the unbalance of several functional classes, and the difficulty of univocally determining negative examples. Moreover, the hierarchical relationships between functional classes that characterize both the Gene Ontology and FunCat taxonomies motivate the development of hierarchy-aware prediction methods that showed significantly better performances than hierarchical-unaware “flat” prediction methods. In this paper, we provide a comprehensive review of hierarchical methods for protein function prediction based on ensembles of learning machines. According to this general approach, a separate learning machine is trained to learn a specific functional term and then the resulting predictions are assembled in a “consensus” ensemble decision, taking into account the hierarchical relationships between classes. The main hierarchical ensemble methods proposed in the literature are discussed in the context of existing computational methods for protein function prediction, highlighting their characteristics, advantages, and limitations. Open problems of this exciting research area of computational biology are finally considered, outlining novel perspectives for future research. PMID:25937954

  14. Upstream ORF affects MYCN translation depending on exon 1b alternative splicing

    PubMed Central

    2009-01-01

    Background The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNΔ1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. Methods Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNΔ1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. Results Both are translated, but higher levels of protein were seen with MYCNΔ1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNΔ1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNΔ1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCNΔ1b mRNA. Conclusions Existence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction. PMID:20017904

  15. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  16. Modification of sorghum proteins for enhanced functionality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum is the third most widely produced crop in the United States (U.S.) and fifth in the world during fiscal year 2006/07(USDA-FAS, 2007). The use of sorghum in foods faces functional and nutritional constraints due, mainly, to the rigidity of the protein bodies. The disruption and modificatio...

  17. Posttranslational Modification Assays on Functional Protein Microarrays.

    PubMed

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng

    2016-10-03

    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  18. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    PubMed

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-10-22

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria.

  19. Experimental and bioinformatic approaches for interrogating protein-protein interactions to determine protein function.

    PubMed

    Droit, Arnaud; Poirier, Guy G; Hunter, Joanna M

    2005-04-01

    An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. One strategy to determine protein function is to identify the protein-protein interactions. The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. A challenge for bioinformatics is to explore this disparate data and to uncover biologically relevant interactions and pathways. In parallel, there is clearly a need for the development of approaches that can predict novel protein-protein interaction networks in silico. Here, we present an overview of different experimental and bioinformatic methods to elucidate protein-protein interactions.

  20. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  1. Functional protein microarrays by electrohydrodynamic jet printing.

    PubMed

    Shigeta, Kazuyo; He, Ying; Sutanto, Erick; Kang, Somi; Le, An-Phong; Nuzzo, Ralph G; Alleyne, Andrew G; Ferreira, Placid M; Lu, Yi; Rogers, John A

    2012-11-20

    This paper reports the use of advanced forms of electrohydrodynamic jet (e-jet) printing for creating micro- and nanoscale patterns of proteins on various surfaces ranging from flat silica substrates to structured plasmonic crystals, suitable for micro/nanoarray analysis and other applications in both fluorescent and plasmonic detection modes. The approaches function well with diverse classes of proteins, including streptavidin, IgG, fibrinogen, and γ-globulin. Detailed study reveals that the printing process does not adversely alter the protein structure or function, as demonstrated in the specific case of streptavidin through measurements of its binding specificity to biotin-modified DNA. Multinozzle printing systems enable several types of proteins (up to four currently) to be patterned on a single substrate, in rapid fashion and with excellent control over spatial dimensions and registration. High-speed, pulsed operational modes allow large-area printing, with narrow statistical distributions of drop size and spacing in patterns that include millions of droplets. The process is also compatible with the structured surfaces of plasmonic crystal substrates to enable detection without fluorescence. These collective characteristics suggest potential utility of e-jet techniques in wide-ranging areas of biotechnology, where its compatibility with various biomaterials and substrates with different topographies and surface chemistries, and ability to form deposits that range from thick films to submonolayer coatings, derive from the remote, noncontacting physical material transfer mode of operation.

  2. Upstream Binding of Idling RNA Polymerase Modulates Transcription Initiation from a Nearby Promoter*

    PubMed Central

    Gerganova, Veneta; Maurer, Sebastian; Stoliar, Liubov; Japaridze, Aleksandre; Dietler, Giovanni; Nasser, William; Kutateladze, Tamara; Travers, Andrew; Muskhelishvili, Georgi

    2015-01-01

    The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter. PMID:25648898

  3. Heterogeneity in Retroviral Nucleocapsid Protein Function

    NASA Astrophysics Data System (ADS)

    Landes, Christy

    2009-03-01

    Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity as compared to that of the HIV-1 NC protein. HTLV-1 NC contains two zinc fingers with each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of HTLV-1 NC protein. The results indicate that HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR)-DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR-DNA. These results suggest that despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart.

  4. The Amyloid Precursor Protein Controls PIKfyve Function.

    PubMed

    Balklava, Zita; Niehage, Christian; Currinn, Heather; Mellor, Laura; Guscott, Benjamin; Poulin, Gino; Hoflack, Bernard; Wassmer, Thomas

    2015-01-01

    While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

  5. Upstream Swimming in Microbiological Flows.

    PubMed

    Mathijssen, Arnold J T M; Shendruk, Tyler N; Yeomans, Julia M; Doostmohammadi, Amin

    2016-01-15

    Interactions between microorganisms and their complex flowing environments are essential in many biological systems. We develop a model for microswimmer dynamics in non-Newtonian Poiseuille flows. We predict that swimmers in shear-thickening (-thinning) fluids migrate upstream more (less) quickly than in Newtonian fluids and demonstrate that viscoelastic normal stress differences reorient swimmers causing them to migrate upstream at the centerline, in contrast to well-known boundary accumulation in quiescent Newtonian fluids. Based on these observations, we suggest a sorting mechanism to select microbes by swimming speed.

  6. Upstream Swimming in Microbiological Flows

    NASA Astrophysics Data System (ADS)

    Mathijssen, Arnold J. T. M.; Shendruk, Tyler N.; Yeomans, Julia M.; Doostmohammadi, Amin

    2016-01-01

    Interactions between microorganisms and their complex flowing environments are essential in many biological systems. We develop a model for microswimmer dynamics in non-Newtonian Poiseuille flows. We predict that swimmers in shear-thickening (-thinning) fluids migrate upstream more (less) quickly than in Newtonian fluids and demonstrate that viscoelastic normal stress differences reorient swimmers causing them to migrate upstream at the centerline, in contrast to well-known boundary accumulation in quiescent Newtonian fluids. Based on these observations, we suggest a sorting mechanism to select microbes by swimming speed.

  7. Functionalized nanoparticle probes for protein detection

    NASA Astrophysics Data System (ADS)

    Park, Do Hyun; Lee, Jae-Seung

    2015-05-01

    In this Review, we discuss representative studies of recent advances in the development of nanoparticle-based protein detection methods, with a focus on the properties and functionalization of nanoparticle probes, as well as their use in detection schemes. We have focused on functionalized nanoparticle probes because they offer a number of advantages over conventional assays and because their use for detecting protein targets for diagnostic purposed has been demonstrated. In this report, we discuss nanoparticle probes classified by material type (gold, silver, silica, semiconductor, carbon, and virus) and surface functionality (antibody, aptamer, and DNA), which play a critical role in enhancing the sensitivity, selectivity, and efficiency of the detection systems. In particular, the synergistic function of each component of the nanoparticle probe is emphasized in terms of specific chemical and physical properties. This research area is in its early stages with many milestones to reach before nanoparticle probes are successfully applied in the field; however, the substantial ongoing efforts of researchers underline the great promise offered by nanoparticlebased probes for future applications. [Figure not available: see fulltext.

  8. Functional characterization of the alphavirus TF protein.

    PubMed

    Snyder, Jonathan E; Kulcsar, Kirsten A; Schultz, Kimberly L W; Riley, Catherine P; Neary, Jacob T; Marr, Scott; Jose, Joyce; Griffin, Diane E; Kuhn, Richard J

    2013-08-01

    Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

  9. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  10. Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates

    PubMed Central

    Korac, Jelena; Schaeffer, Veronique; Kovacevic, Igor; Clement, Albrecht M.; Jungblut, Benno; Behl, Christian; Terzic, Janos; Dikic, Ivan

    2013-01-01

    SUMMARY Aggregation of misfolded proteins and the associated loss of neurons are considered as a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, Creutzfeld-Jacob disease and Pick’s disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognized various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increase protein aggregation in HeLa cells and morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is co-localized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at position Ser-177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway. PMID:23178947

  11. Multiple functions of microsomal triglyceride transfer protein

    PubMed Central

    2012-01-01

    Microsomal triglyceride transfer protein (MTP) was first identified as a major cellular protein capable of transferring neutral lipids between membrane vesicles. Its role as an essential chaperone for the biosynthesis of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins was established after the realization that abetalipoproteinemia patients carry mutations in the MTTP gene resulting in the loss of its lipid transfer activity. Now it is known that it also plays a role in the biosynthesis of CD1, glycolipid presenting molecules, as well as in the regulation of cholesterol ester biosynthesis. In this review, we will provide a historical perspective about the identification, purification and characterization of MTP, describe methods used to measure its lipid transfer activity, and discuss tissue expression and function. Finally, we will review the role MTP plays in the assembly of apoB-lipoprotein, the regulation of cholesterol ester synthesis, biosynthesis of CD1 proteins and propagation of hepatitis C virus. We will also provide a brief overview about the clinical potentials of MTP inhibition. PMID:22353470

  12. CombFunc: predicting protein function using heterogeneous data sources.

    PubMed

    Wass, Mark N; Barton, Geraint; Sternberg, Michael J E

    2012-07-01

    Only a small fraction of known proteins have been functionally characterized, making protein function prediction essential to propose annotations for uncharacterized proteins. In recent years many function prediction methods have been developed using various sources of biological data from protein sequence and structure to gene expression data. Here we present the CombFunc web server, which makes Gene Ontology (GO)-based protein function predictions. CombFunc incorporates ConFunc, our existing function prediction method, with other approaches for function prediction that use protein sequence, gene expression and protein-protein interaction data. In benchmarking on a set of 1686 proteins CombFunc obtains precision and recall of 0.71 and 0.64 respectively for gene ontology molecular function terms. For biological process GO terms precision of 0.74 and recall of 0.41 is obtained. CombFunc is available at http://www.sbg.bio.ic.ac.uk/combfunc.

  13. Dietary proteins and functional gastrointestinal disorders.

    PubMed

    Boettcher, Erica; Crowe, Sheila E

    2013-05-01

    Food intolerance is a common complaint amongst patients with functional gastrointestinal (GI) disorders (FGIDs), including those with irritable bowel syndrome (IBS), functional dyspepsia, as well as gastroesophageal reflux disease. Although there has been a longstanding interest in the possible role of food allergy in IBS, there are limited data supporting the association. However, the prevalence of food allergy is sufficiently high that patients with FGID may also have food allergies or hypersensitivities. Food intolerances or sensitivities are reactions to foods, which are not due to immunological mechanisms. Lactose intolerance is common in the general population and can mimic symptoms of FGID or coexist with FGID. As discussed in other articles in this series, other carbohydrate intolerances may be responsible for symptom generation in patients with IBS and perhaps other FGIDs. There is a great interest in the role of a major dietary protein, gluten, in the production of symptoms that are very similar to those of patients with celiac disease without the enteropathy that characterizes celiac disease. Emerging research into a syndrome known as nonceliac gluten sensitivity suggests a heterogeneous condition with some features of celiac disease but often categorized as FGIDs, including IBS. This article summarizes the role of dietary proteins in the symptoms and pathophysiology of FGIDs.

  14. Protein tyrosine phosphatases: structure-function relationships.

    PubMed

    Tabernero, Lydia; Aricescu, A Radu; Jones, E Yvonne; Szedlacsek, Stefan E

    2008-03-01

    Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.

  15. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    NASA Astrophysics Data System (ADS)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  16. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

    2002-10-15

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  17. Merlin/NF2 functions upstream of the nuclear E3 ubiquitin ligase CRL4DCAF1 to suppress oncogenic gene expression.

    PubMed

    Cooper, Jonathan; Li, Wei; You, Liru; Schiavon, Gaia; Pepe-Caprio, Angela; Zhou, Lu; Ishii, Ryohei; Giovannini, Marco; Hanemann, C Oliver; Long, Stephen B; Erdjument-Bromage, Hediye; Zhou, Pengbo; Tempst, Paul; Giancotti, Filippo G

    2011-08-23

    Integrin-mediated activation of PAK (p21-activated kinase) causes phosphorylation and inactivation of the FERM (4.1, ezrin, radixin, moesin) domain-containing protein Merlin, which is encoded by the NF2 (neurofibromatosis type 2) tumor suppressor gene. Conversely, cadherin engagement inactivates PAK, thus leading to accumulation of unphosphorylated Merlin. Current models imply that Merlin inhibits cell proliferation by inhibiting mitogenic signaling at or near the plasma membrane. We have recently shown that the unphosphorylated, growth-inhibiting form of Merlin accumulates in the nucleus and binds to the E3 ubiquitin ligase CRL4(DCAF1) to suppress its activity. Depletion of DCAF1 blocks the hyperproliferation caused by inactivation of Merlin. Conversely, expression of a Merlin-insensitive DCAF1 mutant counteracts the antimitogenic effect of Merlin. Expression of Merlin or silencing of DCAF1 in Nf2-deficient cells induce an overlapping, tumor-suppressive program of gene expression. Mutations present in some tumors from NF2 patients disrupt Merlin's ability to interact with or inhibit CRL4(DCAF1). Lastly, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells isolated from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. Current studies are aimed at identifying the substrates and mechanism of action of CRL4(DCAF1) and examining its role in NF2-dependent tumorigenesis in mouse models. We propose that Merlin mediates contact inhibition and suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).

  18. Upstream regulation of mycotoxin biosynthesis.

    PubMed

    Alkhayyat, Fahad; Yu, Jae-Hyuk

    2014-01-01

    Mycotoxins are natural contaminants of food and feed products, posing a substantial health risk to humans and animals throughout the world. A plethora of filamentous fungi has been identified as mycotoxin producers and most of these fungal species belong to the genera Aspergillus, Fusarium, and Penicillium. A number of studies have been conducted to better understand the molecular mechanisms of biosynthesis of key mycotoxins and the regulatory cascades controlling toxigenesis. In many cases, the mycotoxin biosynthetic genes are clustered and regulated by one or more pathway-specific transcription factor(s). In addition, as biosynthesis of many secondary metabolites is coordinated with fungal growth and development, there are a number of upstream regulators affecting biosynthesis of mycotoxins in fungi. This review presents a concise summary of the regulation of mycotoxin biosynthesis, focusing on the roles of the upstream regulatory elements governing biosynthesis of aflatoxin and sterigmatocystin in Aspergillus.

  19. Beneficial effects of soy protein consumption for renal function.

    PubMed

    Anderson, James W

    2008-01-01

    Alterations in dietary protein intake have an important role in prevention and management of several forms of kidney disease. Using soy protein instead of animal protein reduces development of kidney disease in animals. Reducing protein intake preserves kidney function in persons with early diabetic kidney disease. Our clinical observations led us to the soy-protein hypothesis that "substitution of soy protein for animal protein results in less hyperfiltration and glomerular hypertension with resulting protection from diabetic nephropathy." These components of soy protein may lead to the benefits: specific peptides, amino acids, and isoflavones. Substituting soy protein for animal protein usually decreases hyperfiltration in diabetic subjects and may reduce urine albumin excretion. Limited data are available on effects of soy peptides, isoflavones, and other soy components on renal function on renal function in diabetes. Further studies are required to discern the specific benefits of soy protein and its components on renal function in diabetic subjects.

  20. Gene sequence of mouse B-type proline-rich protein MP4. Transcriptional start point and an upstream phylogenetic footprint with ets-like and rel/NFkB-like elements.

    PubMed

    Roberts, S G; Layfield, R; Bannister, A J; McDonald, C J

    1991-12-18

    A mouse genomic B-type proline-rich protein (PRP) cosmid clone was isolated by cDNA hybridisation and mapped, the gene region was subcloned and 3770 bp were sequenced. This gene (MP4) contained three introns and encoded a 1020-nt (nt, nucleotide) mRNA for a PRP precursor 300 amino acids long arranged with 11 imperfect 18-residue proline-rich repeats. The transcriptional start point was determined by S1 nuclease mapping and primer extension to be 26 bp downstream of a TATAA sequence. Sequence comparisons revealed that only two regions from positions -650 bp - -30 bp were highly conserved in all other PRP genes, PRP boxes 1 and 2. Box 1 at positions -112 to -135 contained ets-like and rel/NFkB-like elements and was 74% conserved over 23 bp. Box 2 at positions -33 - -51 was 53% conserved over 19 bp. A search of the EMBL and GenBank sequence libraries indicated that PRP box 1 was only present upstream of the known mammalian PRP gene sequences and was absent from other genes. These conserved sequences may thus be relevant to the tissue-specific and beta-adrenergic regulation of PRP gene transcription.

  1. Characterization of the functional properties of carob germ proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins from the carob germ were identified as having gluten-like proteins in 1935. While some biochemical characterization of carob germ proteins and their functionality has been carried out, relatively little has been done when compared to proteins such as gluten. Carob germ proteins were separ...

  2. CombFunc: predicting protein function using heterogeneous data sources

    PubMed Central

    Wass, Mark N.; Barton, Geraint; Sternberg, Michael J. E.

    2012-01-01

    Only a small fraction of known proteins have been functionally characterized, making protein function prediction essential to propose annotations for uncharacterized proteins. In recent years many function prediction methods have been developed using various sources of biological data from protein sequence and structure to gene expression data. Here we present the CombFunc web server, which makes Gene Ontology (GO)-based protein function predictions. CombFunc incorporates ConFunc, our existing function prediction method, with other approaches for function prediction that use protein sequence, gene expression and protein–protein interaction data. In benchmarking on a set of 1686 proteins CombFunc obtains precision and recall of 0.71 and 0.64 respectively for gene ontology molecular function terms. For biological process GO terms precision of 0.74 and recall of 0.41 is obtained. CombFunc is available at http://www.sbg.bio.ic.ac.uk/combfunc. PMID:22641853

  3. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  4. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  5. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  6. How special is the biochemical function of native proteins?

    PubMed

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2016-01-01

    Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation.

  7. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  8. Photo Control of Protein Function Using Photoactive Yellow Protein.

    PubMed

    Reis, Jakeb M; Woolley, G Andrew

    2016-01-01

    Photoswitchable proteins are becoming increasingly common tools for manipulating cellular processes with high spatial and temporal precision. Photoactive yellow protein (PYP) is a small, water-soluble protein that undergoes a blue light induced change in conformation. It can serve as a scaffold for designing new tools to manipulate biological processes, but with respect to other protein scaffolds it presents some technical challenges. Here, we present practical information on how to overcome these, including how to synthesize the PYP chromophore, how to express and purify PYP, and how to screen for desired activity.

  9. Integrated protein function prediction by mining function associations, sequences, and protein–protein and gene–gene interaction networks

    PubMed Central

    Cao, Renzhi; Cheng, Jianlin

    2016-01-01

    Motivations Protein function prediction is an important and challenging problem in bioinformatics and computational biology. Functionally relevant biological information such as protein sequences, gene expression, and protein–protein interactions has been used mostly separately for protein function prediction. One of the major challenges is how to effectively integrate multiple sources of both traditional and new information such as spatial gene–gene interaction networks generated from chromosomal conformation data together to improve protein function prediction. Results In this work, we developed three different probabilistic scores (MIS, SEQ, and NET score) to combine protein sequence, function associations, and protein–protein interaction and spatial gene–gene interaction networks for protein function prediction. The MIS score is mainly generated from homologous proteins found by PSI-BLAST search, and also association rules between Gene Ontology terms, which are learned by mining the Swiss-Prot database. The SEQ score is generated from protein sequences. The NET score is generated from protein–protein interaction and spatial gene–gene interaction networks. These three scores were combined in a new Statistical Multiple Integrative Scoring System (SMISS) to predict protein function. We tested SMISS on the data set of 2011 Critical Assessment of Function Annotation (CAFA). The method performed substantially better than three base-line methods and an advanced method based on protein profile–sequence comparison, profile–profile comparison, and domain co-occurrence networks according to the maximum F-measure. PMID:26370280

  10. Coiled Coil Domain-containing Protein 56 (CCDC56) Is a Novel Mitochondrial Protein Essential for Cytochrome c Oxidase Function*

    PubMed Central

    Peralta, Susana; Clemente, Paula; Sánchez-Martínez, Álvaro; Calleja, Manuel; Hernández-Sierra, Rosana; Matsushima, Yuichi; Adán, Cristina; Ugalde, Cristina; Fernández-Moreno, Miguel Ángel; Kaguni, Laurie S.; Garesse, Rafael

    2012-01-01

    In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5′-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56D6 and ccdc56D11 alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly. PMID:22610097

  11. Nonsymmetric Two-Body Score Function for Protein Fold Recognition:

    NASA Astrophysics Data System (ADS)

    Heo, Muyoung; Cheon, Mookyung; Chang, Iksoo

    The usual two-body score (energy) function to recognize native folds of proteins is Miyazawa-Jernigan (MJ) pairwise-contact function. The pairwise-contact parameters between two amino acids in MJ function are symmetric in a sense that a directional order of amino acids sequence along the backbone of a protein is ignored in constructing score parameters. Here we report that we succeeded in constructing a nonsymmetric two-body score function, capturing a directional order of amino acids sequence, by a perceptron learning and a protein threading. We considered pairs of two adjacent amino acids that are separated by two consecutive peptide bonds with the backbone directionality from the N-terminus to the C-terminus of a protein. We also considered the local environmental character, such as the secondary structures and the hydrophobicity (solvation), of amino acids in protein structures. The score is a corresponding propensity for a directional alignment of these two adjacent amino acids with their local environments. The resulting score function simultaneously recognized native folds of 1006 proteins covering all representative proteins with a homology less than 30% among them. The quality of this score function was validated by a threading test of new distinct 382 proteins with a homology less than 90% among them, and it entailed a high success ratio for recognizing native folds of 364 (95.3%) proteins. It showed a good feasibility of designing protein score functions for protein fold recognition by a perceptron learning and a protein threading.

  12. Computational approaches for rational design of proteins with novel functionalities

    PubMed Central

    Tiwari, Manish Kumar; Singh, Ranjitha; Singh, Raushan Kumar; Kim, In-Won; Lee, Jung-Kul

    2012-01-01

    Proteins are the most multifaceted macromolecules in living systems and have various important functions, including structural, catalytic, sensory, and regulatory functions. Rational design of enzymes is a great challenge to our understanding of protein structure and physical chemistry and has numerous potential applications. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. The field of de novo protein design, although only a few decades old, is beginning to produce exciting results. Developments in this field are already having a significant impact on biotechnology and chemical biology. The application of powerful computational methods for functional protein designing has recently succeeded at engineering target activities. Here, we review recently reported de novo functional proteins that were developed using various protein design approaches, including rational design, computational optimization, and selection from combinatorial libraries, highlighting recent advances and successes. PMID:24688643

  13. Functional innovation from changes in protein domains and their combinations.

    PubMed

    Lees, Jonathan G; Dawson, Natalie L; Sillitoe, Ian; Orengo, Christine A

    2016-06-01

    Domains are the functional building blocks of proteins. In this work we discuss how domains can contribute to the evolution of new functions. Domains themselves can evolve through various mechanisms, altering their intrinsic function. Domains can also facilitate functional innovations by combining with other domains to make novel proteins. We discuss the mechanisms by which domain and domain combinations support functional innovations. We highlight interesting examples where changes in domain combination promote changes at the domain level.

  14. The functional importance of co-evolving residues in proteins.

    PubMed

    Sandler, Inga; Zigdon, Nitzan; Levy, Efrat; Aharoni, Amir

    2014-02-01

    Computational approaches for detecting co-evolution in proteins allow for the identification of protein-protein interaction networks in different organisms and the assignment of function to under-explored proteins. The detection of co-variation of amino acids within or between proteins, moreover, allows for the discovery of residue-residue contacts and highlights functional residues that can affect the binding affinity, catalytic activity, or substrate specificity of a protein. To explore the functional impact of co-evolutionary changes in proteins, a combined experimental and computational approach must be recruited. Here, we review recent studies that apply computational and experimental tools to obtain novel insight into the structure, function, and evolution of proteins. Specifically, we describe the application of co-evolutionary analysis for predicting high-resolution three-dimensional structures of proteins. In addition, we describe computational approaches followed by experimental analysis for identifying specificity-determining residues in proteins. Finally, we discuss studies addressing the importance of such residues in terms of the functional divergence of proteins, allowing proteins to evolve new functions while avoiding crosstalk with existing cellular pathways or forming reproductive barriers and hence promoting speciation.

  15. A Statistical Model of Protein Sequence Similarity and Function Similarity Reveals Overly-Specific Function Predictions

    PubMed Central

    Kolker, Eugene

    2009-01-01

    Background Predicting protein function from primary sequence is an important open problem in modern biology. Not only are there many thousands of proteins of unknown function, current approaches for predicting function must be improved upon. One problem in particular is overly-specific function predictions which we address here with a new statistical model of the relationship between protein sequence similarity and protein function similarity. Methodology Our statistical model is based on sets of proteins with experimentally validated functions and numeric measures of function specificity and function similarity derived from the Gene Ontology. The model predicts the similarity of function between two proteins given their amino acid sequence similarity measured by statistics from the BLAST sequence alignment algorithm. A novel aspect of our model is that it predicts the degree of function similarity shared between two proteins over a continuous range of sequence similarity, facilitating prediction of function with an appropriate level of specificity. Significance Our model shows nearly exact function similarity for proteins with high sequence similarity (bit score >244.7, e-value >1e−62, non-redundant NCBI protein database (NRDB)) and only small likelihood of specific function match for proteins with low sequence similarity (bit score <54.6, e-value <1e−05, NRDB). For sequence similarity ranges in between our annotation model shows an increasing relationship between function similarity and sequence similarity, but with considerable variability. We applied the model to a large set of proteins of unknown function, and predicted functions for thousands of these proteins ranging from general to very specific. We also applied the model to a data set of proteins with previously assigned, specific functions that were electronically based. We show that, on average, these prior function predictions are more specific (quite possibly overly-specific) compared to

  16. Chemical synthesis and biological function of lipidated proteins.

    PubMed

    Yang, Aimin; Zhao, Lei; Wu, Yao-Wen

    2015-01-01

    Lipidated proteins play a key role in many essential biological processes in eukaryotic cells, including signal transduction, membrane trafficking, immune response and pathology. The investigation of the function of lipidated proteins requires access to a reasonable amount of homogenous lipid-modified proteins with defined structures and functional groups. Chemical approaches have provided useful tools to perform such studies. In this review we summarize synthetic methods of lipidated peptides and developments in the chemoselective ligation for the production of lipidated proteins. We introduce the biology of lipidated proteins and highlight the application of synthetic lipidated proteins to tackle important biological questions.

  17. Function and regulation of Rnd proteins.

    PubMed

    Chardin, Pierre

    2006-01-01

    The Rnd proteins, which form a distinct sub-group of the Rho family of small GTP-binding proteins, have been shown to regulate the organization of the actin cytoskeleton in several tissues. In the brain, they participate in neurite extension, whereas in smooth muscle, they modulate contractility. Recent evidence has shown that Rnd3 (RhoE) is also involved in the regulation of cell-cycle progression and transformation, indicating that these proteins might have other, as yet unexplored roles.

  18. Functional Proteins from Short Peptides: Dayhoff's Hypothesis Turns 50.

    PubMed

    Romero Romero, M Luisa; Rabin, Avigayel; Tawfik, Dan S

    2016-12-23

    First and foremost: Margaret Dayhoff's 1966 hypothesis on the origin of proteins is now an accepted model for the emergence of large, globular, functional proteins from short, simple peptides. However, the fundamental question of how the first protein(s) emerged still stands. The tools and hypotheses pioneered by Dayhoff, and the over 65 million protein sequences and 12 000 structures known today, enable those who follow in her footsteps to address this question.

  19. Functional properties of select edible oilseed proteins.

    PubMed

    Sharma, Girdhari M; Su, Mengna; Joshi, Aditya U; Roux, Kenneth H; Sathe, Shridhar K

    2010-05-12

    Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16.

  20. Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

    PubMed

    Jaiswal, Mamta; Dvorsky, Radovan; Ahmadian, Mohammad Reza

    2013-02-08

    The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

  1. Under-folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis.

    PubMed

    Uversky, Vladimir N

    2013-11-01

    For decades, protein function was intimately linked to the presence of a unique, aperiodic crystal-like structure in a functional protein. The two only places for conformational ensembles of under-folded (or partially folded) protein forms in this picture were either the end points of the protein denaturation processes or transiently populated folding intermediates. Recent years witnessed dramatic change in this perception and conformational ensembles, which the under-folded proteins are, have moved from the shadow. Accumulated to date data suggest that a protein can exist in at least three global forms-functional and folded, functional and intrinsically disordered (nonfolded), and nonfunctional and misfolded/aggregated. Under-folded protein states are crucial for each of these forms, serving as important folding intermediates of ordered proteins, or as functional states of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), or as pathology triggers of misfolded proteins. Based on these observations, conformational ensembles of under-folded proteins can be classified as transient (folding and misfolding intermediates) and permanent (IDPs and stable misfolded proteins). Permanently under-folded proteins can further be split into intentionally designed (IDPs and IDPRs) and unintentionally designed (misfolded proteins). Although intrinsic flexibility, dynamics, and pliability are crucial for all under-folded proteins, the different categories of under-foldedness are differently encoded in protein amino acid sequences.

  2. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  3. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  4. Recent approaches in physical modification of protein functionality.

    PubMed

    Mirmoghtadaie, Leila; Shojaee Aliabadi, Saeedeh; Hosseini, Seyede Marzieh

    2016-05-15

    Today, there is a growing demand for novel technologies, such as high hydrostatic pressure, irradiation, ultrasound, filtration, supercritical carbon dioxide, plasma technology, and electrical methods, which are not based on chemicals or heat treatment for modifying ingredient functionality and extending product shelf life. Proteins are essential components in many food processes, and provide various functions in food quality and stability. They can create interfacial films that stabilize emulsions and foams as well as interact to make networks that play key roles in gel and edible film production. These properties of protein are referred to as 'protein functionality', because they can be modified by different processing. The common protein modification (chemical, enzymatic and physical) methods have strong effects on the structure and functionality of food proteins. Furthermore, novel technologies can modify protein structure and functional properties that will be reviewed in this study.

  5. Method for printing functional protein microarrays

    NASA Technical Reports Server (NTRS)

    Delehanty, James B.; Ligler, Frances S.

    2003-01-01

    Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

  6. A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice.

    PubMed

    Sheng, Peike; Wu, Fuqing; Tan, Junjie; Zhang, Huan; Ma, Weiwei; Chen, Liping; Wang, Jiachang; Wang, Jie; Zhu, Shanshan; Guo, Xiuping; Wang, Jiulin; Zhang, Xin; Cheng, Zhijun; Bao, Yiqun; Wu, Chuanyin; Liu, Xuanming; Wan, Jianmin

    2016-09-01

    Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.

  7. Effect of the quality of the interaction data on predicting protein function from protein-protein interactions.

    PubMed

    Ni, Qing-Shan; Wang, Zheng-Zhi; Li, Gang-Guo; Wang, Guang-Yun; Zhao, Ying-Jie

    2009-03-01

    Protein function prediction is an important issue in the post-genomic era. When protein function is deduced from protein interaction data, the traditional methods treat each interaction sample equally, where the qualities of the interaction samples are seldom taken into account. In this paper, we investigate the effect of the quality of protein-protein interaction data on predicting protein function. Moreover, two improved methods, weight neighbour counting method (WNC) and weight chi-square method (WCHI), are proposed by considering the quality of interaction samples with the neighbour counting method (NC) and chi-square method (CHI). Experimental results have shown that the qualities of interaction samples affect the performances of protein function prediction methods seriously. It is also demonstrated that WNC and WCHI methods outperform NC and CHI methods in protein function prediction when example weights are chosen properly.

  8. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  9. Structure and function of antifreeze proteins.

    PubMed Central

    Davies, Peter L; Baardsnes, Jason; Kuiper, Michael J; Walker, Virginia K

    2002-01-01

    High-resolution three-dimensional structures are now available for four of seven non-homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been defined by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic -- more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts. PMID:12171656

  10. Bio-basis function neural networks in protein data mining.

    PubMed

    Yang, Zheng Rong; Hamer, Rebecca

    2007-01-01

    Accurately identifying functional sites in proteins is one of the most important topics in bioinformatics and systems biology. In bioinformatics, identifying protease cleavage sites in protein sequences can aid drug/inhibitor design. In systems biology, post-translational protein-protein interaction activity is one of the major components for analyzing signaling pathway activities. Determining functional sites using laboratory experiments are normally time consuming and expensive. Computer programs have therefore been widely used for this kind of task. Mining protein sequence data using computer programs covers two major issues: 1) discovering how amino acid specificity affects functional sites and 2) discovering what amino acid specificity is. Both need a proper coding mechanism prior to using a proper machine learning algorithm. The development of the bio-basis function neural network (BBFNN) has made a new way for protein sequence data mining. The bio-basis function used in BBFNN is biologically sound in well coding biological information in protein sequences, i.e. well measuring the similarity between protein sequences. BBFNN has therefore been outperforming conventional neural networks in many subjects of protein sequence data mining from protease cleavage site prediction to disordered protein identification. This review focuses on the variants of BBFNN and their applications in mining protein sequence data.

  11. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

  12. [Extranuclear functions of protein sumoylation in the central nervous system].

    PubMed

    Martin, Stéphane

    2009-01-01

    Post-translational protein modifications play essential roles in many aspects of cellular functions and therefore in the maintenance of cell integrity. These protein modifications are involved at all stages of neuronal communication within the central nervous system. Sumoylation is a reversible post-translational protein modification that consists in the covalent labelling of a small protein called SUMO to lysine residues of selected target proteins. Sumoylation is a well characterized regulator of nuclear functions and has recently emerged as a key factor for numerous extranuclear processes. Furthermore, sumoylation has recently been shown to modulate synaptic transmission and is also implicated in a wide range of neurodegenerative diseases.

  13. Internal symmetry in protein structures: prevalence, functional relevance and evolution.

    PubMed

    Balaji, Santhanam

    2015-06-01

    Symmetry has been found at various levels of biological organization in the protein structural universe. Numerous evolutionary studies have proposed connections between internal symmetry within protein tertiary structures, quaternary associations and protein functions. Recent computational methods, such as SymD and CE-Symm, facilitate a large-scale detection of internal symmetry in protein structures. Based on the results from these methods, about 20% of SCOP folds, superfamilies and families are estimated to have structures with internal symmetry (Figure 1d). All-β and membrane proteins fold classes contain a relatively high number of unique instances of internal symmetry. In addition to the axis of symmetry, anecdotal evidence suggests that, the region of connection or contact between symmetric units could coincide with functionally relevant sites within a fold. General principles that underlie protein internal symmetry and their connections to protein structural integrity and functions remain to be elucidated.

  14. Structure-based Methods for Computational Protein Functional Site Prediction

    PubMed Central

    Dukka, B KC

    2013-01-01

    Due to the advent of high throughput sequencing techniques and structural genomic projects, the number of gene and protein sequences has been ever increasing. Computational methods to annotate these genes and proteins are even more indispensable. Proteins are important macromolecules and study of the function of proteins is an important problem in structural bioinformatics. This paper discusses a number of methods to predict protein functional site especially focusing on protein ligand binding site prediction. Initially, a short overview is presented on recent advances in methods for selection of homologous sequences. Furthermore, a few recent structural based approaches and sequence-and-structure based approaches for protein functional sites are discussed in details. PMID:24688745

  15. Alkylation damage by lipid electrophiles targets functional protein systems.

    PubMed

    Codreanu, Simona G; Ullery, Jody C; Zhu, Jing; Tallman, Keri A; Beavers, William N; Porter, Ned A; Marnett, Lawrence J; Zhang, Bing; Liebler, Daniel C

    2014-03-01

    Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

  16. A large-scale evaluation of computational protein function prediction.

    PubMed

    Radivojac, Predrag; Clark, Wyatt T; Oron, Tal Ronnen; Schnoes, Alexandra M; Wittkop, Tobias; Sokolov, Artem; Graim, Kiley; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa; Pandey, Gaurav; Yunes, Jeffrey M; Talwalkar, Ameet S; Repo, Susanna; Souza, Michael L; Piovesan, Damiano; Casadio, Rita; Wang, Zheng; Cheng, Jianlin; Fang, Hai; Gough, Julian; Koskinen, Patrik; Törönen, Petri; Nokso-Koivisto, Jussi; Holm, Liisa; Cozzetto, Domenico; Buchan, Daniel W A; Bryson, Kevin; Jones, David T; Limaye, Bhakti; Inamdar, Harshal; Datta, Avik; Manjari, Sunitha K; Joshi, Rajendra; Chitale, Meghana; Kihara, Daisuke; Lisewski, Andreas M; Erdin, Serkan; Venner, Eric; Lichtarge, Olivier; Rentzsch, Robert; Yang, Haixuan; Romero, Alfonso E; Bhat, Prajwal; Paccanaro, Alberto; Hamp, Tobias; Kaßner, Rebecca; Seemayer, Stefan; Vicedo, Esmeralda; Schaefer, Christian; Achten, Dominik; Auer, Florian; Boehm, Ariane; Braun, Tatjana; Hecht, Maximilian; Heron, Mark; Hönigschmid, Peter; Hopf, Thomas A; Kaufmann, Stefanie; Kiening, Michael; Krompass, Denis; Landerer, Cedric; Mahlich, Yannick; Roos, Manfred; Björne, Jari; Salakoski, Tapio; Wong, Andrew; Shatkay, Hagit; Gatzmann, Fanny; Sommer, Ingolf; Wass, Mark N; Sternberg, Michael J E; Škunca, Nives; Supek, Fran; Bošnjak, Matko; Panov, Panče; Džeroski, Sašo; Šmuc, Tomislav; Kourmpetis, Yiannis A I; van Dijk, Aalt D J; ter Braak, Cajo J F; Zhou, Yuanpeng; Gong, Qingtian; Dong, Xinran; Tian, Weidong; Falda, Marco; Fontana, Paolo; Lavezzo, Enrico; Di Camillo, Barbara; Toppo, Stefano; Lan, Liang; Djuric, Nemanja; Guo, Yuhong; Vucetic, Slobodan; Bairoch, Amos; Linial, Michal; Babbitt, Patricia C; Brenner, Steven E; Orengo, Christine; Rost, Burkhard; Mooney, Sean D; Friedberg, Iddo

    2013-03-01

    Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.

  17. A large-scale evaluation of computational protein function prediction

    PubMed Central

    Radivojac, Predrag; Clark, Wyatt T; Ronnen Oron, Tal; Schnoes, Alexandra M; Wittkop, Tobias; Sokolov, Artem; Graim, Kiley; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa; Pandey, Gaurav; Yunes, Jeffrey M; Talwalkar, Ameet S; Repo, Susanna; Souza, Michael L; Piovesan, Damiano; Casadio, Rita; Wang, Zheng; Cheng, Jianlin; Fang, Hai; Gough, Julian; Koskinen, Patrik; Törönen, Petri; Nokso-Koivisto, Jussi; Holm, Liisa; Cozzetto, Domenico; Buchan, Daniel W A; Bryson, Kevin; Jones, David T; Limaye, Bhakti; Inamdar, Harshal; Datta, Avik; Manjari, Sunitha K; Joshi, Rajendra; Chitale, Meghana; Kihara, Daisuke; Lisewski, Andreas M; Erdin, Serkan; Venner, Eric; Lichtarge, Olivier; Rentzsch, Robert; Yang, Haixuan; Romero, Alfonso E; Bhat, Prajwal; Paccanaro, Alberto; Hamp, Tobias; Kassner, Rebecca; Seemayer, Stefan; Vicedo, Esmeralda; Schaefer, Christian; Achten, Dominik; Auer, Florian; Böhm, Ariane; Braun, Tatjana; Hecht, Maximilian; Heron, Mark; Hönigschmid, Peter; Hopf, Thomas; Kaufmann, Stefanie; Kiening, Michael; Krompass, Denis; Landerer, Cedric; Mahlich, Yannick; Roos, Manfred; Björne, Jari; Salakoski, Tapio; Wong, Andrew; Shatkay, Hagit; Gatzmann, Fanny; Sommer, Ingolf; Wass, Mark N; Sternberg, Michael J E; Škunca, Nives; Supek, Fran; Bošnjak, Matko; Panov, Panče; Džeroski, Sašo; Šmuc, Tomislav; Kourmpetis, Yiannis A I; van Dijk, Aalt D J; ter Braak, Cajo J F; Zhou, Yuanpeng; Gong, Qingtian; Dong, Xinran; Tian, Weidong; Falda, Marco; Fontana, Paolo; Lavezzo, Enrico; Di Camillo, Barbara; Toppo, Stefano; Lan, Liang; Djuric, Nemanja; Guo, Yuhong; Vucetic, Slobodan; Bairoch, Amos; Linial, Michal; Babbitt, Patricia C; Brenner, Steven E; Orengo, Christine; Rost, Burkhard; Mooney, Sean D; Friedberg, Iddo

    2013-01-01

    Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based Critical Assessment of protein Function Annotation (CAFA) experiment. Fifty-four methods representing the state-of-the-art for protein function prediction were evaluated on a target set of 866 proteins from eleven organisms. Two findings stand out: (i) today’s best protein function prediction algorithms significantly outperformed widely-used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is significant need for improvement of currently available tools. PMID:23353650

  18. BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

    PubMed Central

    2012-01-01

    Background Automated function prediction has played a central role in determining the biological functions of bacterial proteins. Typically, protein function annotation relies on homology, and function is inferred from other proteins with similar sequences. This approach has become popular in bacterial genomics because it is one of the few methods that is practical for large datasets and because it does not require additional functional genomics experiments. However, the existing solutions produce erroneous predictions in many cases, especially when query sequences have low levels of identity with the annotated source protein. This problem has created a pressing need for improvements in homology-based annotation. Results We present an automated method for the functional annotation of bacterial protein sequences. Based on sequence similarity searches, BLANNOTATOR accurately annotates query sequences with one-line summary descriptions of protein function. It groups sequences identified by BLAST into subsets according to their annotation and bases its prediction on a set of sequences with consistent functional information. We show the results of BLANNOTATOR's performance in sets of bacterial proteins with known functions. We simulated the annotation process for 3090 SWISS-PROT proteins using a database in its state preceding the functional characterisation of the query protein. For this dataset, our method outperformed the five others that we tested, and the improved performance was maintained even in the absence of highly related sequence hits. We further demonstrate the value of our tool by analysing the putative proteome of Lactobacillus crispatus strain ST1. Conclusions BLANNOTATOR is an accurate method for bacterial protein function prediction. It is practical for genome-scale data and does not require pre-existing sequence clustering; thus, this method suits the needs of bacterial genome and metagenome researchers. The method and a web-server are available at

  19. Small-molecule control of protein function through Staudinger reduction

    NASA Astrophysics Data System (ADS)

    Luo, Ji; Liu, Qingyang; Morihiro, Kunihiko; Deiters, Alexander

    2016-11-01

    Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

  20. Computational design of proteins with novel structure and functions

    NASA Astrophysics Data System (ADS)

    Wei, Yang; Lu-Hua, Lai

    2016-01-01

    Computational design of proteins is a relatively new field, where scientists search the enormous sequence space for sequences that can fold into desired structure and perform desired functions. With the computational approach, proteins can be designed, for example, as regulators of biological processes, novel enzymes, or as biotherapeutics. These approaches not only provide valuable information for understanding of sequence-structure-function relations in proteins, but also hold promise for applications to protein engineering and biomedical research. In this review, we briefly introduce the rationale for computational protein design, then summarize the recent progress in this field, including de novo protein design, enzyme design, and design of protein-protein interactions. Challenges and future prospects of this field are also discussed. Project supported by the National Basic Research Program of China (Grant No. 2015CB910300), the National High Technology Research and Development Program of China (Grant No. 2012AA020308), and the National Natural Science Foundation of China (Grant No. 11021463).

  1. De novo design of functional proteins: Toward artificial hydrogenases.

    PubMed

    Faiella, Marina; Roy, Anindya; Sommer, Dayn; Ghirlanda, Giovanna

    2013-11-01

    Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well-folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

  2. PUF proteins: Cellular functions and potential applications.

    PubMed

    Kiani, Seyed Jalal; Taheri, Tahereh; Rafati, Sima; Samimi-Rad, Katayoun

    2016-09-14

    RNA-binding proteins play critical roles in the regulation of gene expression. Among several families of RNA-binding proteins, PUF (Pumilio and FBF) proteins have been the subject of extensive investigations, as they can bind RNA in a sequence-specific manner and they are evolutionarily conserved among a wide range of organisms. The outstanding feature of these proteins is a highly conserved RNA-binding domain, which is known as the Pumilio-homology domain (PUM-HD) that mostly consists of eight tandem repeats. Each repeat recognizes an RNA base with a simple three-letter code that can be programmed in order to change the sequence-specificity of the protein. Using this tailored architecture, researchers have been able to change the specificity of the PUM-HD and target desired transcripts in the cell, even in subcellular compartments. The potential applications of this versatile tool in molecular cell biology seem unbounded and the use of these factors in pharmaceutics might be an interesting field of study in near future.

  3. Protein Carbonylation and Adipocyte Mitochondrial Function*

    PubMed Central

    Curtis, Jessica M.; Hahn, Wendy S.; Stone, Matthew D.; Inda, Jacob J.; Droullard, David J.; Kuzmicic, Jovan P.; Donoghue, Margaret A.; Long, Eric K.; Armien, Anibal G.; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J.; Bernlohr, David A.

    2012-01-01

    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte. PMID:22822087

  4. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  5. Timing Correlations in Proteins Predict Functional Modules and Dynamic Allostery.

    PubMed

    Lin, Milo M

    2016-04-20

    How protein structure encodes functionality is not fully understood. For example, long-range intraprotein communication can occur without measurable conformational change and is often not captured by existing structural correlation functions. It is shown here that important functional information is encoded in the timing of protein motions, rather than motion itself. I introduce the conditional activity function to quantify such timing correlations among the degrees of freedom within proteins. For three proteins, the conditional activities between side-chain dihedral angles were computed using the output of microseconds-long atomistic simulations. The new approach demonstrates that a sparse fraction of side-chain pairs are dynamically correlated over long distances (spanning protein lengths up to 7 nm), in sharp contrast to structural correlations, which are short-ranged (<1 nm). Regions of high self- and inter-side-chain dynamical correlations are found, corresponding to experimentally determined functional modules and allosteric connections, respectively.

  6. Utilization of alkyne bioconjugations to modulate protein function.

    PubMed

    Maza, Johnathan C; Howard, Christina A; Vipani, Megha A; Travis, Christopher R; Young, Douglas D

    2017-01-01

    The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

  7. A survey of computational intelligence techniques in protein function prediction.

    PubMed

    Tiwari, Arvind Kumar; Srivastava, Rajeev

    2014-01-01

    During the past, there was a massive growth of knowledge of unknown proteins with the advancement of high throughput microarray technologies. Protein function prediction is the most challenging problem in bioinformatics. In the past, the homology based approaches were used to predict the protein function, but they failed when a new protein was different from the previous one. Therefore, to alleviate the problems associated with homology based traditional approaches, numerous computational intelligence techniques have been proposed in the recent past. This paper presents a state-of-the-art comprehensive review of various computational intelligence techniques for protein function predictions using sequence, structure, protein-protein interaction network, and gene expression data used in wide areas of applications such as prediction of DNA and RNA binding sites, subcellular localization, enzyme functions, signal peptides, catalytic residues, nuclear/G-protein coupled receptors, membrane proteins, and pathway analysis from gene expression datasets. This paper also summarizes the result obtained by many researchers to solve these problems by using computational intelligence techniques with appropriate datasets to improve the prediction performance. The summary shows that ensemble classifiers and integration of multiple heterogeneous data are useful for protein function prediction.

  8. Functional assembly of a randomly cleaved protein.

    PubMed Central

    Shiba, K; Schimmel, P

    1992-01-01

    The sequence of a 939-amino acid polypeptide that is a member of the aminoacyl-tRNA synthetase class of enzymes has been aligned with sequences of 15 related proteins. This alignment guided the design of 18 fragment pairs that were tested for internal sequence complementarity by reconstitution of enzyme activity. Reconstitution was achieved with fragments that divide the protein at both nonconserved and conserved sequences, including locations proximal to or within elements believed to form critical elements of secondary structure. Structure assembly is sufficiently flexible to accommodate fusion of short segments of unrelated sequences at fragment junctions. Complementary chain packing interactions and chain flexibility appear to be widely distributed throughout the sequence and are sufficient to reconstruct large three-dimensional structures from an array of disconnected pieces. The results may have implications for the evolution and assembly of large proteins. Images PMID:1542687

  9. Bioinformatics pipeline for functional identification and characterization of proteins

    NASA Astrophysics Data System (ADS)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; Krzywkowski, Tomasz; Świerkula, Katarzyna; PlÄ der, Wojciech; Przybecki, Zbigniew

    2015-09-01

    The new sequencing methods, called Next Generation Sequencing gives an opportunity to possess a vast amount of data in short time. This data requires structural and functional annotation. Functional identification and characterization of predicted proteins could be done by in silico approches, thanks to a numerous computational tools available nowadays. However, there is a need to confirm the results of proteins function prediction using different programs and comparing the results or confirm experimentally. Here we present a bioinformatics pipeline for structural and functional annotation of proteins.

  10. Biases in the experimental annotations of protein function and their effect on our understanding of protein function space.

    PubMed

    Schnoes, Alexandra M; Ream, David C; Thorman, Alexander W; Babbitt, Patricia C; Friedberg, Iddo

    2013-01-01

    The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the "few articles - many proteins" phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments.

  11. A review of protein function prediction under machine learning perspective.

    PubMed

    Bernardes, Juliana S; Pedreira, Carlos E

    2013-08-01

    Protein function prediction is one of the most challenging problems in the post-genomic era. The number of newly identified proteins has been exponentially increasing with the advances of the high-throughput techniques. However, the functional characterization of these new proteins was not incremented in the same proportion. To fill this gap, a large number of computational methods have been proposed in the literature. Early approaches have explored homology relationships to associate known functions to the newly discovered proteins. Nevertheless, these approaches tend to fail when a new protein is considerably different (divergent) from previously known ones. Accordingly, more accurate approaches, that use expressive data representation and explore sophisticate computational techniques are required. Regarding these points, this review provides a comprehensible description of machine learning approaches that are currently applied to protein function prediction problems. We start by defining several problems enrolled in understanding protein function aspects, and describing how machine learning can be applied to these problems. We aim to expose, in a systematical framework, the role of these techniques in protein function inference, sometimes difficult to follow up due to the rapid evolvement of the field. With this purpose in mind, we highlight the most representative contributions, the recent advancements, and provide an insightful categorization and classification of machine learning methods in functional proteomics.

  12. Composition and functional properties of Lupinus campestris protein isolates.

    PubMed

    Rodríguez-Ambriz, S L; Martínez-Ayala, A L; Millán, F; Dávila-Ortíz, G

    2005-09-01

    Protein isolates from L. campestris and soybean seeds were prepared using isoelectric precipitation (PI) and micellization (MI) procedures. The amount of protein recovered was considerably higher with the isoelectric precipitation than with the micellization procedure (60% and 30%, respectively). Protein contents were higher than 90% in protein isolates. Antinutritional factors content (alkaloids, lectins, and tannins) were reduced to innocuous levels after protein isolate preparation. Minimum protein solubility for the precipitated lupin protein isolate (LPI) was at pH 4.0, and between pH 4 and 6 for the micellized lupin protein isolate (LMI), increasing at both extremes of the pH scale. Water absorption for the LMI was 1.3 ml/g of protein and its oil absorption 2.2 ml/g of protein. The LPI had 1.7 ml/g of protein in both water and oil absorption. Foaming capacity and stability was pH-dependent. Foaming capacity was higher at pH 2 and lower near the protein isoelectric points. Minimum protein concentration for gelation in LMI was 8% w/v at pH 4, while for LPI was 6% at pH 4 and 6. Amino acid composition in L. campestris flour and protein isolates was high in lysine and low in methionine. Most of the essential amino acids in lupin protein isolates were at acceptable levels compared to a reference pattern for infants and adults. The electrophoretic pattern of both protein isolates showed three bands with different mobilities, suggesting that the protein fractions belong to alpha-conglutin (11S-like protein), beta-conglutin (7S-like protein) and gamma-conglutin. It is proven that some of the functional properties of L. campestris protein isolates are similar to those soybean protein isolates recovered under equal conditions.

  13. Functional constraints on adaptive evolution of protein ubiquitination sites

    PubMed Central

    Lu, Liang; Li, Yang; Liu, Zhongyang; Liang, Fengji; Guo, Feifei; Yang, Shuai; Wang, Dan; He, Yangzhige; Xiong, Jianghui; Li, Dong; He, Fuchu

    2017-01-01

    It is still unclear whether there exist functional constraints on the evolution of protein ubiquitination sites, because most previous studies regarded all protein ubiquitination sites as a whole or only focused on limited structural properties. We tried to clarify the relation between functional constraints and ubiquitination sites evolution. We investigated the evolutionary conservation of human ubiquitination sites in a broad evolutionary scale from G. gorilla to S. pombe, and we found that in organisms originated after the divergence of vertebrate, ubiquitination sites are more conserved than their flanking regions, while the opposite tendency is observed before this divergence time. By grouping the ubiquitination proteins into different functional categories, we confirm that many functional constraints like certain molecular functions, protein tissue expression specificity and protein connectivity in protein-protein interaction network enhance the evolutionary conservation of ubiquitination sites. Furthermore, by analyzing the gains of ubiquitination sites at different divergence time and their functional characters, we validate that the emergences of ubiquitination sites at different evolutionary time were also affected by the uncovered functional constraints. The above results suggest that functional constraints on the adaptive evolution of ubiquitination sites increase the opportunity for ubiquitination to synthetically regulate various cellular and developmental processes during evolution. PMID:28054638

  14. Housekeeping genes tend to show reduced upstream sequence conservation

    PubMed Central

    Farré, Domènec; Bellora, Nicolás; Mularoni, Loris; Messeguer, Xavier; Albà, M Mar

    2007-01-01

    Background Understanding the constraints that operate in mammalian gene promoter sequences is of key importance to understand the evolution of gene regulatory networks. The level of promoter conservation varies greatly across orthologous genes, denoting differences in the strength of the evolutionary constraints. Here we test the hypothesis that the number of tissues in which a gene is expressed is related in a significant manner to the extent of promoter sequence conservation. Results We show that mammalian housekeeping genes, expressed in all or nearly all tissues, show significantly lower promoter sequence conservation, especially upstream of position -500 with respect to the transcription start site, than genes expressed in a subset of tissues. In addition, we evaluate the effect of gene function, CpG island content and protein evolutionary rate on promoter sequence conservation. Finally, we identify a subset of transcription factors that bind to motifs that are specifically over-represented in housekeeping gene promoters. Conclusion This is the first report that shows that the promoters of housekeeping genes show reduced sequence conservation with respect to genes expressed in a more tissue-restricted manner. This is likely to be related to simpler gene expression, requiring a smaller number of functional cis-regulatory motifs. PMID:17626644

  15. Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

    PubMed Central

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

  16. Protein function prediction using guilty by association from interaction networks.

    PubMed

    Piovesan, Damiano; Giollo, Manuel; Ferrari, Carlo; Tosatto, Silvio C E

    2015-12-01

    Protein function prediction from sequence using the Gene Ontology (GO) classification is useful in many biological problems. It has recently attracted increasing interest, thanks in part to the Critical Assessment of Function Annotation (CAFA) challenge. In this paper, we introduce Guilty by Association on STRING (GAS), a tool to predict protein function exploiting protein-protein interaction networks without sequence similarity. The assumption is that whenever a protein interacts with other proteins, it is part of the same biological process and located in the same cellular compartment. GAS retrieves interaction partners of a query protein from the STRING database and measures enrichment of the associated functional annotations to generate a sorted list of putative functions. A performance evaluation based on CAFA metrics and a fair comparison with optimized BLAST similarity searches is provided. The consensus of GAS and BLAST is shown to improve overall performance. The PPI approach is shown to outperform similarity searches for biological process and cellular compartment GO predictions. Moreover, an analysis of the best practices to exploit protein-protein interaction networks is also provided.

  17. A comparative protein function analysis databaseof different Leishmania strains

    PubMed Central

    Dikhit, Manas Ranjan; Nathasharma, Yangya Prasad; Patel, Lelin; Rana, Sindhu Prava; Sahoo, Ganesh Chandra; Das, Pradeep

    2011-01-01

    A complete understanding of different protein functional families and template information opens new avenues for novel drug development. Protein identification and analysis software performs a central role in the investigation of proteins and leads to the development of refined database for description of proteins of different Leishmania strains. There are certain databases for different strains that lack template information and functional family annotation. Rajendra Memorial Research Institute of Medical Sciences (RMRIMS) has developed a web-based unique database to provide information about functional families of different proteins and its template information in different Leishmania species. Based on the template information users can model the tertiary structure of protein. The database facilitates significant relationship between template information and possible protein functional families assigned to different proteins by SVMProt. This database is designed to provide comprehensive descriptions of certain important proteins found in four different species of Leishmania i.e. L. donovani, L. infantum, L. major and L. braziliensis. A specific characterization information table provides information related to species and specific functional families. This database aims to be a resource for scientists working on proteomics. The database is freely available at http://biomedinformri.org/calp/. PMID:21464840

  18. Roles for text mining in protein function prediction.

    PubMed

    Verspoor, Karin M

    2014-01-01

    The Human Genome Project has provided science with a hugely valuable resource: the blueprints for life; the specification of all of the genes that make up a human. While the genes have all been identified and deciphered, it is proteins that are the workhorses of the human body: they are essential to virtually all cell functions and are the primary mechanism through which biological function is carried out. Hence in order to fully understand what happens at a molecular level in biological organisms, and eventually to enable development of treatments for diseases where some aspect of a biological system goes awry, we must understand the functions of proteins. However, experimental characterization of protein function cannot scale to the vast amount of DNA sequence data now available. Computational protein function prediction has therefore emerged as a problem at the forefront of modern biology (Radivojac et al., Nat Methods 10(13):221-227, 2013).Within the varied approaches to computational protein function prediction that have been explored, there are several that make use of biomedical literature mining. These methods take advantage of information in the published literature to associate specific proteins with specific protein functions. In this chapter, we introduce two main strategies for doing this: association of function terms, represented as Gene Ontology terms (Ashburner et al., Nat Genet 25(1):25-29, 2000), to proteins based on information in published articles, and a paradigm called LEAP-FS (Literature-Enhanced Automated Prediction of Functional Sites) in which literature mining is used to validate the predictions of an orthogonal computational protein function prediction method.

  19. 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.

  20. Protein Structure and Function Prediction Using I-TASSER.

    PubMed

    Yang, Jianyi; Zhang, Yang

    2015-12-17

    I-TASSER is a hierarchical protocol for automated protein structure prediction and structure-based function annotation. Starting from the amino acid sequence of target proteins, I-TASSER first generates full-length atomic structural models from multiple threading alignments and iterative structural assembly simulations followed by atomic-level structure refinement. The biological functions of the protein, including ligand-binding sites, enzyme commission number, and gene ontology terms, are then inferred from known protein function databases based on sequence and structure profile comparisons. I-TASSER is freely available as both an on-line server and a stand-alone package. This unit describes how to use the I-TASSER protocol to generate structure and function prediction and how to interpret the prediction results, as well as alternative approaches for further improving the I-TASSER modeling quality for distant-homologous and multi-domain protein targets.

  1. Collective Dynamics Differentiates Functional Divergence in Protein Evolution

    PubMed Central

    Glembo, Tyler J.; Farrell, Daniel W.; Gerek, Z. Nevin; Thorpe, M. F.; Ozkan, S. Banu

    2012-01-01

    Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function. PMID:22479170

  2. Mammalian protein glycosylation--structure versus function.

    PubMed

    Defaus, S; Gupta, P; Andreu, D; Gutiérrez-Gallego, R

    2014-06-21

    Carbohydrates fulfil many common as well as extremely important functions in nature. They show a variety of molecular displays--e.g., free mono-, oligo-, and polysaccharides, glycolipids, proteoglycans, glycoproteins, etc.--with particular roles and localizations in living organisms. Structure-specific peculiarities are so many and diverse that it becomes virtually impossible to cover them all from an analytical perspective. Hence this manuscript, focused on mammalian glycosylation, rather than a complete list of analytical descriptors or recognized functions for carbohydrate structures, comprehensively reviews three central issues in current glycoscience, namely (i) structural analysis of glycoprotein glycans, covering both classical and novel approaches for teasing out the structural puzzle as well as potential pitfalls of these processes; (ii) an overview of functions attributed to carbohydrates, covering from monosaccharide to complex, well-defined epitopes and full glycans, including post-glycosylational modifications, and (iii) recent technical advances allowing structural identification of glycoprotein glycans with simultaneous assignation of biological functions.

  3. PTPN14 Forms a Complex with Kibra and LATS1 Proteins and Negatively Regulates the YAP Oncogenic Function*

    PubMed Central

    Wilson, Kayla E.; Li, Ying-Wei; Yang, Nuo; Shen, He; Orillion, Ashley R.; Zhang, Jianmin

    2014-01-01

    The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. Pivotal effectors of this pathway are YAP/TAZ, transcriptional co-activators whose dysfunction contributes to epithelial-to-mesenchymal transition and malignant transformation. Therefore, it is of great importance to decipher the mechanisms underlying the regulations of YAP/TAZ at various levels. Here we report that non-receptor tyrosine phosphatase 14 (PTPN14) interacts with the Kibra protein. The interaction between PTPN14 and Kibra is through the PPXY domain of PTPN14 and WW domain of Kibra. PTPN14 and Kibra can induce the LATS1 activation independently and cooperatively. Interestingly, activation of LATS1 by PTPN14 is dependent on the C terminus of PTPN14 and independent of the upstream mammalian STE20-like kinase (MST) proteins. Furthermore, we demonstrate that PTPN14 increases the LAST1 protein stability. Last, overexpression of Kibra rescues the increased cell migration and aberrant three-dimensional morphogenesis induced by knockdown of PTPN14, and this rescue is mediated through the activation of the upstream LATS1 kinase and subsequent cytoplasmic sequestration of YAP. In summary, our results indicate a potential regulatory role of PTPN14 in the Hippo pathway and demonstrate another layer of regulation in the YAP oncogenic function. PMID:25023289

  4. Musashi RNA-binding protein 2 regulates estrogen receptor 1 function in breast cancer.

    PubMed

    Kang, M-H; Jeong, K J; Kim, W Y; Lee, H J; Gong, G; Suh, N; Győrffy, B; Kim, S; Jeong, S-Y; Mills, G B; Park, Y-Y

    2017-03-23

    Musashi RNA-binding protein 2 (MSI2) has important roles in human cancer. However, the regulatory mechanisms by which MSI2 alters breast cancer pathophysiology have not been clearly identified. Here we demonstrate that MSI2 directly regulates estrogen receptor 1 (ESR1), which is a well-known therapeutic target and has been shown to reflect clinical outcomes in breast cancer. Based on gene expression data analysis, we found that MSI2 expression was highly enriched in estrogen receptor (ER)-positive breast cancer and that MSI2 expression was significantly correlated with ESR1 expression, including expression of ESR1 downstream target genes. In addition, MSI2 levels were associated with clinical outcomes. MSI2 influenced breast cancer cell growth by altering ESR1 function. MSI2 alters ESR1 by binding specific sites in ESR1 RNA and by increasing ESR1 protein stability. Taken together, our findings identified a novel regulatory mechanism of MSI2 as an upstream regulator of ESR1 and revealed the clinical relevance of the RNA-binding protein MSI2 in breast cancer.

  5. Function and regulation of Rnd proteins in cortical projection neuron migration

    PubMed Central

    Azzarelli, Roberta; Guillemot, François; Pacary, Emilie

    2015-01-01

    The mammalian cerebral cortex contains a high variety of neuronal subtypes that acquire precise spatial locations and form long or short-range connections to establish functional neuronal circuits. During embryonic development, cortical projection neurons are generated in the areas lining the lateral ventricles and they subsequently undergo radial migration to reach the position of their final maturation within the cortical plate. The control of the neuroblast migratory behavior and the coordination of the migration process with other neurogenic events such as cell cycle exit, differentiation and final maturation are crucial to normal brain development. Among the key regulators of cortical neuron migration, the small GTP binding proteins of the Rho family and the atypical Rnd members play important roles in integrating intracellular signaling pathways into changes in cytoskeletal dynamics and motility behavior. Here we review the role of Rnd proteins during cortical neuronal migration and we discuss both the upstream mechanisms that regulate Rnd protein activity and the downstream molecular pathways that mediate Rnd effects on cell cytoskeleton. PMID:25705175

  6. Maintenance of native-like protein dynamics may not be required for engineering functional proteins.

    PubMed

    Gobeil, Sophie M C; Clouthier, Christopher M; Park, Jaeok; Gagné, Donald; Berghuis, Albert M; Doucet, Nicolas; Pelletier, Joelle N

    2014-10-23

    Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

  7. Understanding the folding-function tradeoff in proteins.

    PubMed

    Gosavi, Shachi

    2013-01-01

    When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the "function-less" folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.

  8. Text Mining Improves Prediction of Protein Functional Sites

    PubMed Central

    Cohn, Judith D.; Ravikumar, Komandur E.

    2012-01-01

    We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

  9. 19 CFR 351.523 - Upstream subsidies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... DUTIES Identification and Measurement of Countervailable Subsidies § 351.523 Upstream subsidies. (a... countervailable subsidy rate on the input product, multiplied by the proportion of the total production costs of...—(1) Presumptions. In evaluating whether an upstream subsidy has a significant effect on the cost...

  10. Design of protein function leaps by directed domain interface evolution

    PubMed Central

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-01-01

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting “affinity clamp” had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution. PMID:18445649

  11. Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.

    PubMed

    Zhou, Huanyu; Dickson, Matthew E; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N

    2015-09-22

    Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique.

  12. Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

    PubMed

    Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-10-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

  13. Protein carbonylation and muscle function in COPD and other conditions.

    PubMed

    Barreiro, Esther

    2014-01-01

    Skeletal muscle, the most abundant tissue in mammals, is essential for any activity in life. Muscle dysfunction is a common systemic manifestation in highly prevalent conditions such as chronic obstructive pulmonary disease (COPD), cancer cachexia, and sepsis. It has a significant impact on exercise tolerance, thus worsening the patients' quality of life and survival. Among several factors, oxidative stress is a major player in the etiology of skeletal muscle dysfunction associated with those conditions. Whereas low levels of oxidants are absolutely required for normal cell adaptation, high levels of reactive oxygen species (ROS) alter the function and structure of molecules such as proteins, DNA, and lipids. Specifically, protein carbonylation, a common variety of protein oxidation, was shown to alter the function of key enzymes and structural proteins involved in muscle contractile performance. Moreover, increased levels of ROS may also activate proteolytic systems, thus leading to enhanced protein breakdown in several models. In the current review, the specific modifications induced by carbonylation in protein structure and function in muscles have been described. Furthermore, the potential role of ROS in the activation of proteolytic systems in skeletal muscles is also discussed. The review summarizes the effects of protein carbonylation on muscles in several models and conditions such as COPD, disuse muscle atrophy, cancer cachexia, sepsis, and aging. Future research should focus on the elucidation of the specific protein sites modified by ROS in these muscles using redox proteomics analyses and on the assessment of the consequent alterations in protein function and stability.

  14. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices.

  15. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  16. Targeting functional motifs of a protein family.

    PubMed

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β-lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β-lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β-lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  17. Accuracy of functional surfaces on comparatively modeled protein structures

    PubMed Central

    Zhao, Jieling; Dundas, Joe; Kachalo, Sema; Ouyang, Zheng; Liang, Jie

    2012-01-01

    Identification and characterization of protein functional surfaces are important for predicting protein function, understanding enzyme mechanism, and docking small compounds to proteins. As the rapid speed of accumulation of protein sequence information far exceeds that of structures, constructing accurate models of protein functional surfaces and identify their key elements become increasingly important. A promising approach is to build comparative models from sequences using known structural templates such as those obtained from structural genome projects. Here we assess how well this approach works in modeling binding surfaces. By systematically building three-dimensional comparative models of proteins using Modeller, we determine how well functional surfaces can be accurately reproduced. We use an alpha shape based pocket algorithm to compute all pockets on the modeled structures, and conduct a large-scale computation of similarity measurements (pocket RMSD and fraction of functional atoms captured) for 26,590 modeled enzyme protein structures. Overall, we find that when the sequence fragment of the binding surfaces has more than 45% identity to that of the tempalte protein, the modeled surfaces have on average an RMSD of 0.5 Å, and contain 48% or more of the binding surface atoms, with nearly all of the important atoms in the signatures of binding pockets captured. PMID:21541664

  18. Upstream Waves and Particles at the Moon

    NASA Astrophysics Data System (ADS)

    Harada, Y.; Halekas, J. S.

    2016-02-01

    This chapter presents an up-to-date catalog of Moon-related particle populations and lunar upstream waves obtained from in situ measurements at low (<˜100 km) and high altitudes, aimed at organizing and clarifying the currently available information on this complex region, where multiple categories of waves and particles coexist. It then briefly outlines the observed properties of a variety of classes of lunar upstream waves, as well as their generation mechanisms currently proposed, in association with the lunar upstream particle distributions. The lunar upstream region magnetically connected to the Moon and its wake, the fore-moon, represents a remarkably rich zoo of different classes of waves and different types of particles. Although recent observations have substantially enhanced our knowledge by revealing a number of new categories of upstream particles and waves at the Moon, many fundamental questions remain unanswered, and these are outlined in the chapter.

  19. Self-Assembled Materials Made from Functional Recombinant Proteins.

    PubMed

    Jang, Yeongseon; Champion, Julie A

    2016-10-18

    Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on

  20. Gene Ontology Function prediction in Mollicutes using Protein-Protein Association Networks

    PubMed Central

    2011-01-01

    Background Many complex systems can be represented and analysed as networks. The recent availability of large-scale datasets, has made it possible to elucidate some of the organisational principles and rules that govern their function, robustness and evolution. However, one of the main limitations in using protein-protein interactions for function prediction is the availability of interaction data, especially for Mollicutes. If we could harness predicted interactions, such as those from a Protein-Protein Association Networks (PPAN), combining several protein-protein network function-inference methods with semantic similarity calculations, the use of protein-protein interactions for functional inference in this species would become more potentially useful. Results In this work we show that using PPAN data combined with other approximations, such as functional module detection, orthology exploitation methods and Gene Ontology (GO)-based information measures helps to predict protein function in Mycoplasma genitalium. Conclusions To our knowledge, the proposed method is the first that combines functional module detection among species, exploiting an orthology procedure and using information theory-based GO semantic similarity in PPAN of the Mycoplasma species. The results of an evaluation show a higher recall than previously reported methods that focused on only one organism network. PMID:21486441

  1. Transposable element fragments in protein-coding regions and their contributions to human functional proteins.

    PubMed

    Wu, Ming; Li, Li; Sun, Zhirong

    2007-10-15

    Transposable elements (TEs) and their contributions to protein-coding regions are of particular interest. Here we searched for TE fragments in Homo sapiens at both the transcript and protein levels. We found evidence in support of TE exonization and its association with alternative splicing. Despite recent findings that long evolutionary times are required to incorporate TE into proteins, we found many functional proteins with translated TE cassettes derived from young TEs. Analyses of two Bcl-family proteins and Alu-encoded segments suggest the coding and functional potential of TE sequences.

  2. Are non-functional, unfolded proteins ('junk proteins') common in the genome?

    PubMed

    Lovell, Simon C

    2003-11-20

    It has recently been shown that many proteins are unfolded in their functional state. In addition, a large number of stretches of protein sequences are predicted to be unfolded. It has been argued that the high frequency of occurrence of these predicted unfolded sequences indicates that the majority of these sequences must also be functional. These sequences tend to be of low complexity. It is well established that certain types of low-complexity sequences are genetically unstable, and are prone to expand in the genome. It is possible, therefore, that in addition to these well-characterised functional unfolded proteins, there are a large number of unfolded proteins that are non-functional. Analogous to 'junk DNA' these protein sequences may arise due to physical characteristics of DNA. Their high frequency may reflect, therefore, the high probability of expansion in the genome. Such 'junk proteins' would not be advantageous, and may be mildly deleterious to the cell.

  3. Elastic properties of protein functionalized nanoporous polymer films

    SciTech Connect

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that protein functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.

  4. Cellular strategies for regulating functional and nonfunctional protein aggregation.

    PubMed

    Gsponer, Jörg; Babu, M Madan

    2012-11-29

    Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier's principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control.

  5. Biochemical functional predictions for protein structures of unknown or uncertain function.

    PubMed

    Mills, Caitlyn L; Beuning, Penny J; Ondrechen, Mary Jo

    2015-01-01

    With the exponential growth in the determination of protein sequences and structures via genome sequencing and structural genomics efforts, there is a growing need for reliable computational methods to determine the biochemical function of these proteins. This paper reviews the efforts to address the challenge of annotating the function at the molecular level of uncharacterized proteins. While sequence- and three-dimensional-structure-based methods for protein function prediction have been reviewed previously, the recent trends in local structure-based methods have received less attention. These local structure-based methods are the primary focus of this review. Computational methods have been developed to predict the residues important for catalysis and the local spatial arrangements of these residues can be used to identify protein function. In addition, the combination of different types of methods can help obtain more information and better predictions of function for proteins of unknown function. Global initiatives, including the Enzyme Function Initiative (EFI), COMputational BRidges to EXperiments (COMBREX), and the Critical Assessment of Function Annotation (CAFA), are evaluating and testing the different approaches to predicting the function of proteins of unknown function. These initiatives and global collaborations will increase the capability and reliability of methods to predict biochemical function computationally and will add substantial value to the current volume of structural genomics data by reducing the number of absent or inaccurate functional annotations.

  6. Biochemical functional predictions for protein structures of unknown or uncertain function

    PubMed Central

    Mills, Caitlyn L.; Beuning, Penny J.; Ondrechen, Mary Jo

    2015-01-01

    With the exponential growth in the determination of protein sequences and structures via genome sequencing and structural genomics efforts, there is a growing need for reliable computational methods to determine the biochemical function of these proteins. This paper reviews the efforts to address the challenge of annotating the function at the molecular level of uncharacterized proteins. While sequence- and three-dimensional-structure-based methods for protein function prediction have been reviewed previously, the recent trends in local structure-based methods have received less attention. These local structure-based methods are the primary focus of this review. Computational methods have been developed to predict the residues important for catalysis and the local spatial arrangements of these residues can be used to identify protein function. In addition, the combination of different types of methods can help obtain more information and better predictions of function for proteins of unknown function. Global initiatives, including the Enzyme Function Initiative (EFI), COMputational BRidges to EXperiments (COMBREX), and the Critical Assessment of Function Annotation (CAFA), are evaluating and testing the different approaches to predicting the function of proteins of unknown function. These initiatives and global collaborations will increase the capability and reliability of methods to predict biochemical function computationally and will add substantial value to the current volume of structural genomics data by reducing the number of absent or inaccurate functional annotations. PMID:25848497

  7. A functional protein retention and release multilayer with high stability

    NASA Astrophysics Data System (ADS)

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-01

    Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by

  8. Evolution: A Guide to Perturb Protein Function and Networks

    PubMed Central

    Lichtarge, Olivier; Wilkins, Angela

    2010-01-01

    Summary Protein interactions give rise to networks that control cell fate in health and disease; selective means to probe these interactions are therefore of wide interest. We discuss here Evolutionary Tracing (ET), a comparative method to identify protein functional sites and to guide experiments that selectively block, recode, or mimic their amino acid determinants. These studies suggest, in principle, a scalable approach to perturb individual links in protein networks. PMID:20444593

  9. Classifying proteins into functional groups based on all-versus-all BLAST of 10 million proteins.

    PubMed

    Kolker, Natali; Higdon, Roger; Broomall, William; Stanberry, Larissa; Welch, Dean; Lu, Wei; Haynes, Winston; Barga, Roger; Kolker, Eugene

    2011-01-01

    To address the monumental challenge of assigning function to millions of sequenced proteins, we completed the first of a kind all-versus-all sequence alignments using BLAST for 9.9 million proteins in the UniRef100 database. Microsoft Windows Azure produced over 3 billion filtered records in 6 days using 475 eight-core virtual machines. Protein classification into functional groups was then performed using Hive and custom jars implemented on top of Apache Hadoop utilizing the MapReduce paradigm. First, using the Clusters of Orthologous Genes (COG) database, a length normalized bit score (LNBS) was determined to be the best similarity measure for classification of proteins. LNBS achieved sensitivity and specificity of 98% each. Second, out of 5.1 million bacterial proteins, about two-thirds were assigned to significantly extended COG groups, encompassing 30 times more assigned proteins. Third, the remaining proteins were classified into protein functional groups using an innovative implementation of a single-linkage algorithm on an in-house Hadoop compute cluster. This implementation significantly reduces the run time for nonindexed queries and optimizes efficient clustering on a large scale. The performance was also verified on Amazon Elastic MapReduce. This clustering assigned nearly 2 million proteins to approximately half a million different functional groups. A similar approach was applied to classify 2.8 million eukaryotic sequences resulting in over 1 million proteins being assign to existing KOG groups and the remainder clustered into 100,000 functional groups.

  10. FunPred-1: protein function prediction from a protein interaction network using neighborhood analysis.

    PubMed

    Saha, Sovan; Chatterjee, Piyali; Basu, Subhadip; Kundu, Mahantapas; Nasipuri, Mita

    2014-12-01

    Proteins are responsible for all biological activities in living organisms. Thanks to genome sequencing projects, large amounts of DNA and protein sequence data are now available, but the biological functions of many proteins are still not annotated in most cases. The unknown function of such non-annotated proteins may be inferred or deduced from their neighbors in a protein interaction network. In this paper, we propose two new methods to predict protein functions based on network neighborhood properties. FunPred 1.1 uses a combination of three simple-yet-effective scoring techniques: the neighborhood ratio, the protein path connectivity and the relative functional similarity. FunPred 1.2 applies a heuristic approach using the edge clustering coefficient to reduce the search space by identifying densely connected neighborhood regions. The overall accuracy achieved in FunPred 1.2 over 8 functional groups involving hetero-interactions in 650 yeast proteins is around 87%, which is higher than the accuracy with FunPred 1.1. It is also higher than the accuracy of many of the state-of-the-art protein function prediction methods described in the literature. The test datasets and the complete source code of the developed software are now freely available at http://code.google.com/p/cmaterbioinfo/ .

  11. Tactile Teaching: Exploring Protein Structure/Function Using Physical Models

    ERIC Educational Resources Information Center

    Herman, Tim; Morris, Jennifer; Colton, Shannon; Batiza, Ann; Patrick, Michael; Franzen, Margaret; Goodsell, David S.

    2006-01-01

    The technology now exists to construct physical models of proteins based on atomic coordinates of solved structures. We review here our recent experiences in using physical models to teach concepts of protein structure and function at both the high school and the undergraduate levels. At the high school level, physical models are used in a…

  12. The HMG-1 box protein family: classification and functional relationships.

    PubMed Central

    Baxevanis, A D; Landsman, D

    1995-01-01

    The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts. Images PMID:7784217

  13. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  14. Discovering Conformational Sub-States Relevant to Protein Function

    PubMed Central

    Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

    2011-01-01

    Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

  15. Versatile multi-functionalization of protein nanofibrils for biosensor applications.

    PubMed

    Sasso, L; Suei, S; Domigan, L; Healy, J; Nock, V; Williams, M A K; Gerrard, J A

    2014-01-01

    Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.

  16. Assessment of protein set coherence using functional annotations

    PubMed Central

    Chagoyen, Monica; Carazo, Jose M; Pascual-Montano, Alberto

    2008-01-01

    Background Analysis of large-scale experimental datasets frequently produces one or more sets of proteins that are subsequently mined for functional interpretation and validation. To this end, a number of computational methods have been devised that rely on the analysis of functional annotations. Although current methods provide valuable information (e.g. significantly enriched annotations, pairwise functional similarities), they do not specifically measure the degree of homogeneity of a protein set. Results In this work we present a method that scores the degree of functional homogeneity, or coherence, of a set of proteins on the basis of the global similarity of their functional annotations. The method uses statistical hypothesis testing to assess the significance of the set in the context of the functional space of a reference set. As such, it can be used as a first step in the validation of sets expected to be homogeneous prior to further functional interpretation. Conclusion We evaluate our method by analysing known biologically relevant sets as well as random ones. The known relevant sets comprise macromolecular complexes, cellular components and pathways described for Saccharomyces cerevisiae, which are mostly significantly coherent. Finally, we illustrate the usefulness of our approach for validating 'functional modules' obtained from computational analysis of protein-protein interaction networks. Matlab code and supplementary data are available at PMID:18937846

  17. Pathways and functions of the Werner syndrome protein.

    PubMed

    Lee, Jae Wan; Harrigan, Jeanine; Opresko, Patricia L; Bohr, Vilhelm A

    2005-01-01

    Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS). WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN protein regarding its binding partners and the site of protein-protein interaction. The mapping analysis of protein interaction sites in WRN for most of its binding partners have revealed a common site of protein-protein interaction in the RecQ conserved (RQC) region of WRN.

  18. CATH FunFHMMer web server: protein functional annotations using functional family assignments.

    PubMed

    Das, Sayoni; Sillitoe, Ian; Lee, David; Lees, Jonathan G; Dawson, Natalie L; Ward, John; Orengo, Christine A

    2015-07-01

    The widening function annotation gap in protein databases and the increasing number and diversity of the proteins being sequenced presents new challenges to protein function prediction methods. Multidomain proteins complicate the protein sequence-structure-function relationship further as new combinations of domains can expand the functional repertoire, creating new proteins and functions. Here, we present the FunFHMMer web server, which provides Gene Ontology (GO) annotations for query protein sequences based on the functional classification of the domain-based CATH-Gene3D resource. Our server also provides valuable information for the prediction of functional sites. The predictive power of FunFHMMer has been validated on a set of 95 proteins where FunFHMMer performs better than BLAST, Pfam and CDD. Recent validation by an independent international competition ranks FunFHMMer as one of the top function prediction methods in predicting GO annotations for both the Biological Process and Molecular Function Ontology. The FunFHMMer web server is available at http://www.cathdb.info/search/by_funfhmmer.

  19. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  20. Using experimental evolution to probe molecular mechanisms of protein function.

    PubMed

    Fischer, Marlies; Kang, Mandeep; Brindle, Nicholas Pj

    2016-02-01

    Directed evolution is a powerful tool for engineering protein function. The process of directed evolution involves iterative rounds of sequence diversification followed by assaying activity of variants and selection. The range of sequence variants and linked activities generated in the course of an evolution are a rich information source for investigating relationships between sequence and function. Key residue positions determining protein function, combinatorial contributors to activity and even potential functional mechanisms have been revealed in directed evolutions. The recent application of high throughput sequencing substantially increases the information that can be retrieved from directed evolution experiments. Combined with computational analysis this additional sequence information has allowed high-resolution analysis of individual residue contributions to activity. These developments promise to significantly enhance the depth of insight that experimental evolution provides into mechanisms of protein function.

  1. Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

    PubMed

    Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

    2013-03-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

  2. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  3. Surfactant-associated proteins: structure, function and clinical implications.

    PubMed

    Ketko, Anastasia K; Donn, Steven M

    2014-01-01

    Surfactant replacement therapy is now the standard of care for infants with respiratory distress syndrome. As the understanding of surfactant structure and function has evolved, surfactant-associated proteins are now understood to be essential components of pulmonary surfactant. Their structural and functional diversity detail the complexity of their contributions to normal pulmonary physiology, and deficiency states result in significant pathology. Engineering synthetic surfactant protein constructs has been a major research focus for replacement therapies. This review highlights what is known about surfactant proteins and how this knowledge is pivotal for future advancements in treating respiratory distress syndrome as well as other pulmonary diseases characterized by surfactant deficiency or inactivation.

  4. Predicting protein functions from redundancies in large-scale protein interaction networks

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj Pratim; Liang, Shoudan

    2003-01-01

    Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

  5. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  6. Sampling Protein Form and Function with the Atomic Force Microscope*

    PubMed Central

    Baclayon, Marian; Roos, Wouter H.; Wuite, Gijs J. L.

    2010-01-01

    To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments. PMID:20562411

  7. Influence of dietary protein on renal function in dogs.

    PubMed

    Bovée, K C

    1991-11-01

    Two previously published studies in dogs with reduced renal function are reviewed. In the first study, renal function and biochemical responses to dietary changes were studied in four dogs with stable chronic renal failure. The objective was to determine if dogs with moderate stable failure adjust to diets with varied protein and electrolyte content. These dogs were found to have the capacity to adapt to a wide range of dietary protein and electrolyte intake. The only exception was found in dogs fed a reduced-protein diet, which failed to appropriately adjust renal tubular excretion of sodium and phosphate. The only advantage of reduced dietary protein in this study was a reduction in blood urea nitrogen (BUN). Disadvantages of reduced-protein diets were reduced glomerular filtration rate (GFR) and renal plasma flow. In the second study, the hypothesis that large amounts of dietary protein sustain renal hyperfunction and produce progressive glomerulosclerosis in dogs as previously reported in rats was tested. Results failed to find a pattern of deterioration of renal function over 4 y. Light microscopic changes and electron microscopy also failed to find glomerular injury similar to that reported in rodents. These results do not support the hypothesis that feeding a high protein diet had a significant adverse effect on renal function or morphology.

  8. How optimization of potential functions affects protein folding.

    PubMed Central

    Hao, M H; Scheraga, H A

    1996-01-01

    The relationship between the optimization of the potential function and the foldability of theoretical protein models is studied based on investigations of a 27-mer cubic-lattice protein model and a more realistic lattice model for the protein crambin. In both the simple and the more complicated systems, optimization of the energy parameters achieves significant improvements in the statistical-mechanical characteristics of the systems and leads to foldable protein models in simulation experiments. The foldability of the protein models is characterized by their statistical-mechanical properties--e.g., by the density of states and by Monte Carlo folding simulations of the models. With optimized energy parameters, a high level of consistency exists among different interactions in the native structures of the protein models, as revealed by a correlation function between the optimized energy parameters and the native structure of the model proteins. The results of this work are relevant to the design of a general potential function for folding proteins by theoretical simulations. PMID:8643516

  9. Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins.

    PubMed

    Maeda, Yusuke; Kinoshita, Taroh

    2011-10-01

    Glycosylphosphatidylinositol (GPI) is a glycolipid that is covalently attached to proteins as a post-translational modification. Such modification leads to the anchoring of the protein to the outer leaflet of the plasma membrane. Proteins that are decorated with GPIs have unique properties in terms of their physical nature. In particular, these proteins tend to accumulate in lipid rafts, which are critical for the functions and trafficking of GPI-anchored proteins (GPI-APs). Recent studies mainly using mutant cells revealed that various structural remodeling reactions occur to GPIs present in GPI-APs as they are transported from the endoplasmic reticulum to the cell surface. This review examines the recent progress describing the mechanisms of structural remodeling of mammalian GPI-anchors, such as inositol deacylation, glycan remodeling and fatty acid remodeling, with particular focus on their trafficking and functions, as well as the pathogenesis involving GPI-APs and their deficiency.

  10. The APOBEC Protein Family: United by Structure, Divergent in Function.

    PubMed

    Salter, Jason D; Bennett, Ryan P; Smith, Harold C

    2016-07-01

    The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

  11. Structure and Function of Nematode RNA-Binding Proteins

    PubMed Central

    Kaymak, Ebru; Wee, L.M.; Ryder, Sean P.

    2010-01-01

    RNA-binding proteins are critical effectors of gene expression. They guide mRNA localization, translation, and stability, and potentially play a role in regulating mRNA synthesis. The structural basis for RNA recognition by RNA-binding proteins is the key to understanding how they target specific transcripts for regulation. Compared to other metazoans, nematode genomes contain a significant expansion in several RNA-binding protein families, including Pumilio-FBF (PUF), TTP-like zinc finger (TZF), and argonaute-like (AGO) proteins. Genetic data suggest that individual members of each family have distinct functions, presumably due to sequence variations that alter RNA binding specificity or protein interaction partners. In this review, we highlight example structures and identify the variable regions that likely contribute to functional divergence in nematodes. PMID:20418095

  12. The APSES family proteins in fungi: Characterizations, evolution and functions.

    PubMed

    Zhao, Yong; Su, Hao; Zhou, Jing; Feng, Huihua; Zhang, Ke-Qin; Yang, Jinkui

    2015-08-01

    The APSES protein family belongs to transcriptional factors of the basic helix-loop-helix (bHLH) class, the originally described members (APSES: Asm1p, Phd1p, Sok2p, Efg1p and StuAp) are used to designate this group of proteins, and they have been identified as key regulators of fungal development and other biological processes. APSES proteins share a highly conserved DNA-binding domain (APSES domain) of about 100 amino acids, whose central domain is predicted to form a typical bHLH structure. Besides APSES domain, several APSES proteins also contain additional domains, such as KilA-N and ankyrin repeats. In recent years, an increasing number of APSES proteins have been identified from diverse fungi, and they involve in numerous biological processes, such as sporulation, cellular differentiation, mycelial growth, secondary metabolism and virulence. Most fungi, including Aspergillus fumigatus, Aspergillus nidulans, Candida albicans, Fusarium graminearum, and Neurospora crassa, contain five APSES proteins. However, Cryptococcus neoformans only contains two APSES proteins, and Saccharomyces cerevisiae contains six APSES proteins. The phylogenetic analysis showed the APSES domains from different fungi were grouped into four clades (A, B, C and D), which is consistent with the result of homologous alignment of APSES domains using DNAman. The roles of APSES proteins in clade C have been studied in detail, while little is known about the roles of other APSES proteins in clades A, B and D. In this review, the biochemical properties and functional domains of APSES proteins are predicted and compared, and the phylogenetic relationship among APSES proteins from various fungi are analyzed based on the APSES domains. Moreover, the functions of APSES proteins in different fungi are summarized and discussed.

  13. Acyl-CoA binding proteins: multiplicity and function.

    PubMed

    Gossett, R E; Frolov, A A; Roths, J B; Behnke, W D; Kier, A B; Schroeder, F

    1996-09-01

    The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

  14. Elastic properties of protein functionalized nanoporous polymer films

    DOE PAGES

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that proteinmore » functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.« less

  15. Determining protein function and interaction from genome analysis

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Thompson, Michael J.; Pellegrini, Matteo; Yeates, Todd O.

    2004-08-03

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  16. Predicting Protein Function via Semantic Integration of Multiple Networks.

    PubMed

    Yu, Guoxian; Fu, Guangyuan; Wang, Jun; Zhu, Hailong

    2016-01-01

    Determining the biological functions of proteins is one of the key challenges in the post-genomic era. The rapidly accumulated large volumes of proteomic and genomic data drives to develop computational models for automatically predicting protein function in large scale. Recent approaches focus on integrating multiple heterogeneous data sources and they often get better results than methods that use single data source alone. In this paper, we investigate how to integrate multiple biological data sources with the biological knowledge, i.e., Gene Ontology (GO), for protein function prediction. We propose a method, called SimNet, to Semantically integrate multiple functional association Networks derived from heterogenous data sources. SimNet firstly utilizes GO annotations of proteins to capture the semantic similarity between proteins and introduces a semantic kernel based on the similarity. Next, SimNet constructs a composite network, obtained as a weighted summation of individual networks, and aligns the network with the kernel to get the weights assigned to individual networks. Then, it applies a network-based classifier on the composite network to predict protein function. Experiment results on heterogenous proteomic data sources of Yeast, Human, Mouse, and Fly show that, SimNet not only achieves better (or comparable) results than other related competitive approaches, but also takes much less time. The Matlab codes of SimNet are available at https://sites.google.com/site/guoxian85/simnet.

  17. Protein Conformational Populations and Functionally Relevant Sub-states

    SciTech Connect

    Agarwal, Pratul K; Burger, Virginia; Savol, Andrej; Ramanathan, Arvind; Chennubhotla, Chakra

    2013-01-01

    Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of the protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow

  18. The Protein Information Resource: an integrated public resource of functional annotation of proteins

    PubMed Central

    Wu, Cathy H.; Huang, Hongzhan; Arminski, Leslie; Castro-Alvear, Jorge; Chen, Yongxing; Hu, Zhang-Zhi; Ledley, Robert S.; Lewis, Kali C.; Mewes, Hans-Werner; Orcutt, Bruce C.; Suzek, Baris E.; Tsugita, Akira; Vinayaka, C. R.; Yeh, Lai-Su L.; Zhang, Jian; Barker, Winona C.

    2002-01-01

    The Protein Information Resource (PIR) serves as an integrated public resource of functional annotation of protein data to support genomic/proteomic research and scientific discovery. The PIR, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the PIR-International Protein Sequence Database (PSD), the major annotated protein sequence database in the public domain, containing about 250 000 proteins. To improve protein annotation and the coverage of experimentally validated data, a bibliography submission system is developed for scientists to submit, categorize and retrieve literature information. Comprehensive protein information is available from iProClass, which includes family classification at the superfamily, domain and motif levels, structural and functional features of proteins, as well as cross-references to over 40 biological databases. To provide timely and comprehensive protein data with source attribution, we have introduced a non-redundant reference protein database, PIR-NREF. The database consists of about 800 000 proteins collected from PIR-PSD, SWISS-PROT, TrEMBL, GenPept, RefSeq and PDB, with composite protein names and literature data. To promote database interoperability, we provide XML data distribution and open database schema, and adopt common ontologies. The PIR web site (http://pir.georgetown.edu/) features data mining and sequence analysis tools for information retrieval and functional identification of proteins based on both sequence and annotation information. The PIR databases and other files are also available by FTP (ftp://nbrfa.georgetown.edu/pir_databases). PMID:11752247

  19. ESG: extended similarity group method for automated protein function prediction

    PubMed Central

    Chitale, Meghana; Hawkins, Troy; Park, Changsoon; Kihara, Daisuke

    2009-01-01

    Motivation: Importance of accurate automatic protein function prediction is ever increasing in the face of a large number of newly sequenced genomes and proteomics data that are awaiting biological interpretation. Conventional methods have focused on high sequence similarity-based annotation transfer which relies on the concept of homology. However, many cases have been reported that simple transfer of function from top hits of a homology search causes erroneous annotation. New methods are required to handle the sequence similarity in a more robust way to combine together signals from strongly and weakly similar proteins for effectively predicting function for unknown proteins with high reliability. Results: We present the extended similarity group (ESG) method, which performs iterative sequence database searches and annotates a query sequence with Gene Ontology terms. Each annotation is assigned with probability based on its relative similarity score with the multiple-level neighbors in the protein similarity graph. We will depict how the statistical framework of ESG improves the prediction accuracy by iteratively taking into account the neighborhood of query protein in the sequence similarity space. ESG outperforms conventional PSI-BLAST and the protein function prediction (PFP) algorithm. It is found that the iterative search is effective in capturing multiple-domains in a query protein, enabling accurately predicting several functions which originate from different domains. Availability: ESG web server is available for automated protein function prediction at http://dragon.bio.purdue.edu/ESG/ Contact: cspark@cau.ac.kr; dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19435743

  20. Structure and function of WD40 domain proteins.

    PubMed

    Xu, Chao; Min, Jinrong

    2011-03-01

    The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.

  1. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function.

    PubMed

    Valli, Minoska; Tatto, Nadine E; Peymann, Armin; Gruber, Clemens; Landes, Nils; Ekker, Heinz; Thallinger, Gerhard G; Mattanovich, Diethard; Gasser, Brigitte; Graf, Alexandra B

    2016-09-01

    As manually curated and non-automated BLAST analysis of the published Pichia pastoris genome sequences revealed many differences between the gene annotations of the strains GS115 and CBS7435, RNA-Seq analysis, supported by proteomics, was performed to improve the genome annotation. Detailed analysis of sequence alignment and protein domain predictions were made to extend the functional genome annotation to all P. pastoris sequences. This allowed the identification of 492 new ORFs, 4916 hypothetical UTRs and the correction of 341 incorrect ORF predictions, which were mainly due to the presence of upstream ATG or erroneous intron predictions. Moreover, 175 previously erroneously annotated ORFs need to be removed from the annotation. In total, we have annotated 5325 ORFs. Regarding the functionality of those genes, we improved all gene and protein descriptions. Thereby, the percentage of ORFs with functional annotation was increased from 48% to 73%. Furthermore, we defined functional groups, covering 25 biological cellular processes of interest, by grouping all genes that are part of the defined process. All data are presented in the newly launched genome browser and database available at www.pichiagenome.org In summary, we present a wide spectrum of curation of the P. pastoris genome annotation from gene level to protein function.

  2. Integration of latex protein sequence data provides comprehensive functional overview of latex proteins.

    PubMed

    Cho, Won Kyong; Jo, Yeonhwa; Chu, Hyosub; Park, Sang-Ho; Kim, Kook-Hyung

    2014-03-01

    The laticiferous system is one of the most important conduit systems in higher plants, which produces a milky-like sap known as latex. Latex contains diverse secondary metabolites with various ecological functions. To obtain a comprehensive overview of the latex proteome, we integrated available latex proteins sequences and constructed a comprehensive dataset composed of 1,208 non-redundant latex proteins from 20 various latex-bearing plants. The results of functional analyses revealed that latex proteins are involved in various biological processes, including transcription, translation, protein degradation and the plant response to environmental stimuli. The results of the comparative analysis showed that the functions of the latex proteins are similar to those of phloem, suggesting the functional conservation of plant vascular proteins. The presence of latex proteins in mitochondria and plastids suggests the production of diverse secondary metabolites. Furthermore, using a BLAST search, we identified 854 homologous latex proteins in eight plant species, including three latex-bearing plants, such as papaya, caster bean and cassava, suggesting that latex proteins were newly evolved in vascular plants. Taken together, this study is the largest and most comprehensive in silico analysis of the latex proteome. The results obtained here provide useful resources and information for characterizing the evolution of the latex proteome.

  3. Structural and functional properties of hemp seed protein products.

    PubMed

    Malomo, Sunday A; He, Rong; Aluko, Rotimi E

    2014-08-01

    The effects of pH and protein concentration on some structural and functional properties of hemp seed protein isolate (HPI, 84.15% protein content) and defatted hemp seed protein meal (HPM, 44.32% protein content) were determined. The HPI had minimum protein solubility (PS) at pH 4.0, which increased as pH was decreased or increased. In contrast, the HPM had minimum PS at pH 3.0, which increased at higher pH values. Gel electrophoresis showed that some of the high molecular weight proteins (>45 kDa) present in HPM were not well extracted by the alkali and were absent or present in low ratio in the HPI polypeptide profile. The amino acid composition showed that the isolation process increased the Arg/Lys ratio of HPI (5.52%) when compared to HPM (3.35%). Intrinsic fluorescence and circular dichroism data indicate that the HPI proteins had a well-defined structure at pH 3.0, which was lost as pH value increased. The differences in structural conformation of HPI at different pH values were reflected as better foaming capacity at pH 3.0 when compared to pH 5.0, 7.0, and 9.0. At 10 and 25 mg/mL protein concentrations, emulsions formed by the HPM had smaller oil droplet sizes (higher quality), when compared to the HPI-formed emulsions. In contrast at 50 mg/mL protein concentration, the HPI-formed emulsions had smaller oil droplet sizes (except at pH 3.0). We conclude that the functional properties of hemp seed protein products are dependent on structural conformations as well as protein concentration and pH.

  4. Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

    PubMed

    Jain, Shruti; Bhattacharyya, Kausik; Bakshi, Rachit; Narang, Ankita; Brahmachari, Vani

    2017-04-01

    The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.

  5. Evolutionary Trace Annotation of Protein Function in the Structural Proteome

    PubMed Central

    Erdin, Serkan; Ward, R. Matthew; Venner, Eric

    2010-01-01

    By design, structural genomics (SG) solves many structures that cannot be assigned function based on homology to known proteins. Alternative function annotation methods are therefore needed and this study focuses on function prediction with three-dimensional (3D) templates: small structural motifs built of just a few functionally critical residues. Although experimentally proven functional residues are scarce, we show here that Evolutionary Trace (ET) rankings of residue importance are sufficient to build 3D templates, match them, and then assign Gene Ontology (GO) functions in enzymes and non-enzymes alike. In a high specificity mode, this Evolutionary Trace Annotation (ETA) method covered half (53%) of the 2384 annotated SG protein controls. Three-quarters (76%) of predictions were both correct and complete. The positive predictive value for all GO depths (all-depth PPV) was 84%, and it rose to 94% over GO depths 1– 3 (depth 3 PPV). In a high sensitivity mode coverage rose significantly (84%) while accuracy fell moderately: 68% of predictions were both correct and complete, all-depth PPV was 75%, and depth 3 PPV was 86%. These data concur with prior mutational experiments showing that ET rank information identifies key functional determinants in proteins. In practice, ETA predicted functions in 42% of 3461 un-annotated SG proteins. In 529 cases—including 280 non-enzymes and 21 for metal ion ligands—the expected accuracy is 84% at any GO depth and 94% down to GO depth 3, while for the remaining 931 the expected accuracies are 60% and 71%, respectively. Thus local structural comparisons of evolutionarily important residues can help decipher protein functions to known reliability levels and without prior assumption on functional mechanisms. ETA is available at http://mammoth.bcm.tmc.edu/eta. PMID:20036248

  6. Proteins: sequence to structure and function--current status.

    PubMed

    Shenoy, Sandhya R; Jayaram, B

    2010-11-01

    In an era that has been dominated by Structural Biology for the last 30-40 years, a dramatic change of focus towards sequence analysis has spurred the advent of the genome projects and the resultant diverging sequence/structure deficit. The central challenge of Computational Structural Biology is therefore to rationalize the mass of sequence information into biochemical and biophysical knowledge and to decipher the structural, functional and evolutionary clues encoded in the language of biological sequences. In investigating the meaning of sequences, two distinct analytical themes have emerged: in the first approach, pattern recognition techniques are used to detect similarity between sequences and hence to infer related structures and functions; in the second ab initio prediction methods are used to deduce 3D structure, and ultimately to infer function, directly from the linear sequence. In this article, we attempt to provide a critical assessment of what one may and may not expect from the biological sequences and to identify major issues yet to be resolved. The presentation is organized under several subtitles like protein sequences, pattern recognition techniques, protein tertiary structure prediction, membrane protein bioinformatics, human proteome, protein-protein interactions, metabolic networks, potential drug targets based on simple sequence properties, disordered proteins, the sequence-structure relationship and chemical logic of protein sequences.

  7. Cellular functions of gamma-secretase-related proteins.

    PubMed

    Haffner, Christof; Haass, Christian

    2006-01-01

    Amyloid-beta peptide (Abeta) is generated by gamma-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from gamma-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway.

  8. Conformational diversity analysis reveals three functional mechanisms in proteins

    PubMed Central

    Fornasari, María Silvina

    2017-01-01

    Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call “rigid” (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions. PMID:28192432

  9. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  10. Gene3D: modelling protein structure, function and evolution.

    PubMed

    Yeats, Corin; Maibaum, Michael; Marsden, Russell; Dibley, Mark; Lee, David; Addou, Sarah; Orengo, Christine A

    2006-01-01

    The Gene3D release 4 database and web portal (http://cathwww.biochem.ucl.ac.uk:8080/Gene3D) provide a combined structural, functional and evolutionary view of the protein world. It is focussed on providing structural annotation for protein sequences without structural representatives--including the complete proteome sets of over 240 different species. The protein sequences have also been clustered into whole-chain families so as to aid functional prediction. The structural annotation is generated using HMM models based on the CATH domain families; CATH is a repository for manually deduced protein domains. Amongst the changes from the last publication are: the addition of over 100 genomes and the UniProt sequence database, domain data from Pfam, metabolic pathway and functional data from COGs, KEGG and GO, and protein-protein interaction data from MINT and BIND. The website has been rebuilt to allow more sophisticated querying and the data returned is presented in a clearer format with greater functionality. Furthermore, all data can be downloaded in a simple XML format, allowing users to carry out complex investigations at their own computers.

  11. Metrnl: a secreted protein with new emerging functions

    PubMed Central

    Zheng, Si-li; Li, Zhi-yong; Song, Jie; Liu, Jian-min; Miao, Chao-yu

    2016-01-01

    Secreted proteins play critical roles in physiological and pathological processes and can be used as biomarkers and therapies for aging and disease. Metrnl is a novel secreted protein homologous to the neurotrophin Metrn. But this protein, unlike Metrn that is mainly expressed in the brain, shows a relatively wider distribution in the body with high levels of expression in white adipose tissue and barrier tissues. This protein plays important roles in neural development, white adipose browning and insulin sensitization. Based on its expression and distinct functions, this protein is also called Cometin, Subfatin and Interleukin 39, which refer to its neurotrophic effect, adipokine function and the possible action as a cytokine, respectively. The spectrum of Metrnl functions remains to be determined, and the mechanisms of Metrnl action need to be elucidated. In this review, we focus on the discovery, structural characteristics, expression pattern and physiological functions of Metrnl, which will assist in developing this protein as a new therapeutic target or agent. PMID:27063217

  12. Computational approaches for inferring the functions of intrinsically disordered proteins

    PubMed Central

    Varadi, Mihaly; Vranken, Wim; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular processes and often implicated in human pathological conditions. The critical biological roles of these proteins, despite not adopting a well-defined fold, encouraged structural biologists to revisit their views on the protein structure-function paradigm. Unfortunately, investigating the characteristics and describing the structural behavior of IDPs is far from trivial, and inferring the function(s) of a disordered protein region remains a major challenge. Computational methods have proven particularly relevant for studying IDPs: on the sequence level their dependence on distinct characteristics determined by the local amino acid context makes sequence-based prediction algorithms viable and reliable tools for large scale analyses, while on the structure level the in silico integration of fundamentally different experimental data types is essential to describe the behavior of a flexible protein chain. Here, we offer an overview of the latest developments and computational techniques that aim to uncover how protein function is connected to intrinsic disorder. PMID:26301226

  13. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone.

    PubMed

    Modisakeng, Keoagile W; Jiwaji, Meesbah; Pesce, Eva-Rachele; Robert, Jacques; Amemiya, Chris T; Dorrington, Rosemary A; Blatch, Gregory L

    2009-08-01

    Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

  14. Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera.

    PubMed

    Kumar, Suresh

    2015-01-01

    Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO2 assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency.

  15. Structural Basis of Protein Kinase C Isoform Function

    PubMed Central

    STEINBERG, SUSAN F.

    2010-01-01

    Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells. PMID:18923184

  16. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  17. Functional characterization of AMP-activated protein kinase signaling in tumorigenesis.

    PubMed

    Cheng, Ji; Zhang, Tao; Ji, Hongbin; Tao, Kaixiong; Guo, Jianping; Wei, Wenyi

    2016-12-01

    AMP-activated protein kinase (AMPK) is a ubiquitously expressed metabolic sensor among various species. Specifically, cellular AMPK is phosphorylated and activated under certain stressful conditions, such as energy deprivation, in turn to activate diversified downstream substrates to modulate the adaptive changes and maintain metabolic homeostasis. Recently, emerging evidences have implicated the potential roles of AMPK signaling in tumor initiation and progression. Nevertheless, a comprehensive description on such topic is still in scarcity, especially in combination of its biochemical features with mouse modeling results to elucidate the physiological role of AMPK signaling in tumorigenesis. Hence, we performed this thorough review by summarizing the tumorigenic role of each component along the AMPK signaling, comprising of both its upstream and downstream effectors. Moreover, their functional interplay with the AMPK heterotrimer and exclusive efficacies in carcinogenesis were chiefly explained among genetically altered mice models. Importantly, the pharmaceutical investigations of AMPK relevant medications have also been highlighted. In summary, in this review, we not only elucidate the potential functions of AMPK signaling pathway in governing tumorigenesis, but also potentiate the future targeted strategy aiming for better treatment of aberrant metabolism-associated diseases, including cancer.

  18. A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions.

    PubMed

    Peng, Yingjie; Zeng, Wenwen; Ye, Hui; Han, Kyung Ho; Dharmarajan, Venkatasubramanian; Novick, Scott; Wilson, Ian A; Griffin, Patrick R; Friedman, Jeffrey M; Lerner, Richard A

    2015-08-20

    A major goal of modern protein chemistry is to create new proteins with different functions. One approach is to amalgamate secondary and tertiary structures from different proteins. This is difficult for several reasons, not the least of which is the fact that the junctions between secondary and tertiary structures are not degenerate and usually affect the function and folding of the entire complex. Here, we offer a solution to this problem by coupling a large combinatorial library of about 10(7) different N- and C-terminal junctions to a powerful system that selects for function. Using this approach, the entire Leptin and follicle-stimulating hormone (FSH) were inserted into an antibody. Complexes with full retention of function in vivo and in vitro, although rare, were found easily by using an autocrine selection system to search for hormonal activity. Such large diversity systems, when coupled to robust selection systems, should enable construction of novel therapeutic proteins.

  19. TIA-1 Is a Functional Prion-Like Protein.

    PubMed

    Rayman, Joseph B; Kandel, Eric R

    2016-12-21

    Prions are self-propagating protein conformations that are traditionally regarded as agents of neurodegenerative disease in animals. However, it has become evident that prion-like aggregation of endogenous proteins can also occur under normal physiological conditions (e.g., during memory storage or activation of the immune response). In this review, we focus on the functional prion-related protein TIA-1, an RNA-binding protein that is involved in multiple aspects of RNA metabolism but is best understood in terms of its role in stress granule assembly during the cellular stress response. We propose that stress granule formation provides a useful conceptual framework with which to address the positive role of TIA-1 prion-like aggregation. Elucidating the function of TIA-1 prion-like aggregation will advance our understanding of how prion-based molecular switches are used in normal physiological settings.

  20. Versatile multi-functionalization of protein nanofibrils for biosensor applications

    NASA Astrophysics Data System (ADS)

    Sasso, L.; Suei, S.; Domigan, L.; Healy, J.; Nock, V.; Williams, M. A. K.; Gerrard, J. A.

    2014-01-01

    Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the

  1. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties.

    PubMed

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications.

  2. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties

    PubMed Central

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P.; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications. PMID:26840533

  3. The SARS coronavirus nucleocapsid protein--forms and functions.

    PubMed

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses."

  4. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data.

  5. Structure and function of contractile proteins in muscle fibres.

    PubMed

    Barden, J A; Bennetts, B H; dos Remedios, C G; Hambly, B D; Miki, M; Phillips, L

    1988-01-01

    The structural unit of muscle has long been defined as the myofibril, a supramolecular assembly of a dozen or more proteins of which two, actin and myosin, comprise more than 75%. In the past 40 years since Albert Szent-Gyorgyi first described the contractile response from the complex of actin and myosin, knowledge of the structure and function of these contractile proteins has been substantially refined. This paper describes these new discoveries and identifies the problems which remain to be elucidated.

  6. [Study of molecular function of proteins in human immunodeficiency virus].

    PubMed

    Fujita, Mikako

    2013-01-01

    Human immunodeficiency virus (HIV) has no more than nine genes expressing approximately twenty proteins. When T lymphocytes and macrophages in a body are infected with HIV, these proteins work in turn at specific time and location, causing acquired immunodeficiency syndrome (AIDS), a disease yet to be overcome. Since the elucidation of molecular mechanism of HIV proteins should lead to remedy of AIDS, the author has been engaged in the study of HIV protein in the past decade. Described herein are viral protein X (Vpx), uniquely found in HIV-2, and its homologous protein Vpr found both in HIV-1 and -2. We found that Vpx enhances genome nuclear import in T lymphocytes, and is critical for reverse transcription of viral RNA in macrophages. This finding on the function in macrophages corrected long-term misleading belief. Furthermore, functional region mapping of Vpx was performed. In 2011, the protein SAMHD1 was identified as the host restriction factor counteracted by Vpx, by foreign researchers. After that, our independent study demonstrated the presence of SAMHD1-independent functions of Vpx in T cells, in addition to its SAMHD1-dependent functions in macrophages. Another topic of this review is Gag protein. Recently, it has reported by overseas researchers that PI(4,5)P2 (one of phosphoinositide) regulates Pr55(Gag) localization and assembly. In this study, we determined the binding affinity between N-terminal MA domain of Pr55(Gag) and various phosphoinositide derivatives using surface plasmon resonance. The results suggested that both negatively charged inositol phosphates and hydrophobic acyl chain are required for the MA binding.

  7. Specific protein homeostatic functions of small heat-shock proteins increase lifespan.

    PubMed

    Vos, Michel J; Carra, Serena; Kanon, Bart; Bosveld, Floris; Klauke, Karin; Sibon, Ody C M; Kampinga, Harm H

    2016-04-01

    During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.

  8. Twenty years of protein interaction studies for biological function deciphering.

    PubMed

    Legrain, Pierre; Rain, Jean-Christophe

    2014-07-31

    Intensive methodological developments and technology innovation have been devoted to protein-protein interaction studies over 20years. Genetic indirect assays and sophisticated large scale biochemical analyses have jointly contributed to the elucidation of protein-protein interactions, still with a lot of drawbacks despite heavy investment in human resources and technologies. With the most recent developments in mass spectrometry and computational tools for studying protein content of complex samples, the initial goal of deciphering molecular bases of biological functions is now within reach. Here, we described the various steps of this process and gave examples of key milestones in this scientific story line. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

  9. Organizing protein-DNA hybrids as nanostructures with programmed functionalities.

    PubMed

    Teller, Carsten; Willner, Itamar

    2010-12-01

    The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.

  10. Exceptional overproduction of a functional human membrane protein.

    PubMed

    Nyblom, Maria; Oberg, Fredrik; Lindkvist-Petersson, Karin; Hallgren, Karin; Findlay, Heather; Wikström, Jennie; Karlsson, Anders; Hansson, Orjan; Booth, Paula J; Bill, Roslyn M; Neutze, Richard; Hedfalk, Kristina

    2007-11-01

    Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.

  11. Diversity and functions of protein glycosylation in insects.

    PubMed

    Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

    2017-04-01

    The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.

  12. Functional conservation of an ancestral Pellino protein in helminth species

    PubMed Central

    Cluxton, Christopher D.; Caffrey, Brian E.; Kinsella, Gemma K.; Moynagh, Paul N.; Fares, Mario A.; Fallon, Padraic G.

    2015-01-01

    The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans. PMID:26120048

  13. RACK1, A multifaceted scaffolding protein: Structure and function

    PubMed Central

    2011-01-01

    The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease. PMID:21978545

  14. Functionality of alternative protein in gluten-free product development.

    PubMed

    Deora, Navneet Singh; Deswal, Aastha; Mishra, Hari Niwas

    2015-07-01

    Celiac disease is an immune-mediated disease triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The current treatment for celiac disease is life-long adherence to a strict gluten-exclusion diet. The replacement of gluten presents a significant technological challenge, as it is an essential structure-building protein, which is necessary for formulating high-quality baked goods. A major limitation in the production of gluten-free products is the lack of protein functionality in non-wheat cereals. Additionally, commercial gluten-free mixes usually contain only carbohydrates, which may significantly limit the amount of protein in the diet. In the recent past, various approaches are attempted to incorporate protein-based ingredients and to modify the functional properties for gluten-free product development. This review aims to the highlight functionality of the alternative protein-based ingredients, which can be utilized for gluten-free product development both functionally as well as nutritionally.

  15. SM30 protein function during sea urchin larval spicule formation.

    PubMed

    Wilt, Fred; Killian, Christopher E; Croker, Lindsay; Hamilton, Patricia

    2013-08-01

    A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.

  16. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  17. Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast.

    PubMed Central

    Dorsman, J C; van Heeswijk, W C; Grivell, L A

    1988-01-01

    Two abundant factors, GFI and GFII which interact with the 5' flanking regions of nuclear genes coding for proteins of the mitochondrial respiratory chain have been identified. In one case (subunit VIII of QH2: cytochrome c oxidoreductase) the binding sites for both factors overlap completely and their binding is mutually exclusive. For the other 5' regions tested the GFI and GFII binding sites do not coincide. Interestingly, binding sites for GFI and GFII are also present in or at the 3' ends of the coding regions of two genes of the PHO gene family and in DNA elements important for optimal ARS and CEN function respectively. The sites recognized by GFI conform to the consensus RTCRNNNNNNACGNR, while those recognized by GFII contain the element RTCACGTG. We speculate that GFI and GFII may play a role in different cellular processes, dependent on the context of their binding sites and that one of these processes may be the coordination of the expression of genes involved in mitochondrial biogenesis with the progress of the cell cycle. Images PMID:3045755

  18. Protein-protein interactions in intracellular Ca2+-release channel function.

    PubMed Central

    MacKrill, J J

    1999-01-01

    Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4, 5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein-protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein-protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are 'customized' by the accessory proteins present. PMID:9895277

  19. Optimizing an emperical scoring function for transmembrane protein structure determination.

    SciTech Connect

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  20. SitesIdentify: a protein functional site prediction tool

    PubMed Central

    2009-01-01

    Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify), based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/ PMID:19922660

  1. Comparison of functional properties of 34% and 80% whey protein and milk serum protein concentrates.

    PubMed

    Luck, P J; Vardhanabhuti, B; Yong, Y H; Laundon, T; Barbano, D M; Foegeding, E A

    2013-09-01

    This study compared the functional properties of serum protein concentrate (SPC) with whey protein concentrate (WPC) made from the same milk and with commercial WPC. The experimental SPC and WPC were produced at 34% or 80% protein from the same lot of milk. Protein contents of WPC and SPC were comparable; however, fat content was much lower in SPC compared with WPC and commercial WPC. The effect of drying methods (freeze vs. spray drying) was studied for 34% WPC and SPC. Few differences due to drying method were found in turbidity and gelation; however, drying method made a large difference in foam formation for WPC but not SPC. Between pH 3 and 7, SPC was found to have lower turbidity than WPC; however, protein solubility was similar between SPC and WPC. Foaming and gelation properties of SPC were better than those of WPC. Differences in functional properties may be explained by differences in composition and extent of denaturation or aggregation.

  2. Bactericidal/permeability increasing protein: a multifaceted protein with functions beyond LPS neutralization.

    PubMed

    Balakrishnan, Arjun; Marathe, Sandhya A; Joglekar, Madhura; Chakravortty, Dipshikha

    2013-01-01

    Bactericidal permeability increasing protein (BPI), a 55-60 kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

  3. A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

    PubMed

    Nuth, Manunya; Guan, Hancheng; Ricciardi, Robert P

    2016-12-30

    Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the inner core of the protein. Our mutational analysis revealed that a tripeptide motif ((215)GFI(217)) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly(215) that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that (215)GFI(217) anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.

  4. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  5. Using computational biophysics to understand protein evolution and function

    NASA Astrophysics Data System (ADS)

    Ytreberg, F. Marty

    2010-10-01

    Understanding how proteins evolve and function is vital for human health (e.g., developing better drugs, predicting the outbreak of disease, etc.). In spite of its importance, little is known about the underlying molecular mechanisms behind these biological processes. Computational biophysics has emerged as a useful tool in this area due to its unique ability to obtain a detailed, atomistic view of proteins and how they interact. I will give two examples from our studies where computational biophysics has provided valuable insight: (i) Protein evolution in viruses. Our results suggest that the amino acid changes that occur during high temperature evolution of a virus decrease the binding free energy of the capsid, i.e., these changes increase capsid stability. (ii) Determining realistic structural ensembles for intrinsically disordered proteins. Most methods for determining protein structure rely on the protein folding into a single conformation, and thus are not suitable for disordered proteins. I will describe a new approach that combines experiment and simulation to generate structures for disordered proteins.

  6. Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.

    PubMed

    Behal, Robert H; Betleja, Ewelina; Cole, Douglas G

    2009-01-01

    Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.

  7. A combinatorial scoring function for protein-RNA docking.

    PubMed

    Zhang, Zhao; Lu, Lin; Zhang, Yue; Hua Li, Chun; Wang, Cun Xin; Zhang, Xiao Yi; Tan, Jian Jun

    2017-04-01

    Protein-RNA docking is still an open question. One of the main challenges is to develop an effective scoring function that can discriminate near-native structures from the incorrect ones. To solve the problem, we have constructed a knowledge-based residue-nucleotide pairwise potential with secondary structure information considered for nonribosomal protein-RNA docking. Here we developed a weighted combined scoring function RpveScore that consists of the pairwise potential and six physics-based energy terms. The weights were optimized using the multiple linear regression method by fitting the scoring function to L_rmsd for the bound docking decoys from Benchmark II. The scoring functions were tested on 35 unbound docking cases. The results show that the scoring function RpveScore including all terms performs best. Also RpveScore was compared with the statistical mechanics-based method derived potential ITScore-PR, and the united atom-based statistical potentials QUASI-RNP and DARS-RNP. The success rate of RpveScore is 71.6% for the top 1000 structures and the number of cases where a near-native structure is ranked in top 30 is 25 out of 35 cases. For 32 systems (91.4%), RpveScore can find the binding mode in top 5 that has no lower than 50% native interface residues on protein and nucleotides on RNA. Additionally, it was found that the long-range electrostatic attractive energy plays an important role in distinguishing near-native structures from the incorrect ones. This work can be helpful for the development of protein-RNA docking methods and for the understanding of protein-RNA interactions. RpveScore program is available to the public at http://life.bjut.edu.cn/kxyj/kycg/2017116/14845362285362368_1.html Proteins 2017; 85:741-752. © 2016 Wiley Periodicals, Inc.

  8. Rho1 GTPase and PKC ortholog Pck1 are upstream activators of the cell integrity MAPK pathway in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.

  9. Deciphering fine molecular details of proteins' structure and function with a Protein Surface Topography (PST) method.

    PubMed

    Koromyslova, Anna D; Chugunov, Anton O; Efremov, Roman G

    2014-04-28

    Molecular surfaces are the key players in biomolecular recognition and interactions. Nowadays, it is trivial to visualize a molecular surface and surface-distributed properties in three-dimensional space. However, such a representation trends to be biased and ambiguous in case of thorough analysis. We present a new method to create 2D spherical projection maps of entire protein surfaces and manipulate with them--protein surface topography (PST). It permits visualization and thoughtful analysis of surface properties. PST helps to easily portray conformational transitions, analyze proteins' properties and their dynamic behavior, improve docking performance, and reveal common patterns and dissimilarities in molecular surfaces of related bioactive peptides. This paper describes basic usage of PST with an example of small G-proteins conformational transitions, mapping of caspase-1 intersubunit interface, and intrinsic "complementarity" in the conotoxin-acetylcholine binding protein complex. We suggest that PST is a beneficial approach for structure-function studies of bioactive peptides and small proteins.

  10. Functional and Structural Analysis of the Conserved EFhd2 Protein

    PubMed Central

    Acosta, Yancy Ferrer; Rodríguez Cruz, Eva N.; Vaquer, Ana del C.; Vega, Irving E.

    2013-01-01

    EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2’s calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2’s thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2’s thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins. PMID:22973849

  11. Identification of giant Mimivirus protein functions using RNA interference.

    PubMed

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases.

  12. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.

  13. From protein structure to function via single crystal optical spectroscopy

    PubMed Central

    Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Storici, Paola; Mozzarelli, Andrea

    2015-01-01

    The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms. PMID:25988179

  14. Identification of giant Mimivirus protein functions using RNA interference

    PubMed Central

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases. PMID:25972846

  15. Protein engineering of Cas9 for enhanced function.

    PubMed

    Oakes, Benjamin L; Nadler, Dana C; Savage, David F

    2014-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

  16. Proteins of the corneal stroma: importance in visual function.

    PubMed

    Xuan, Meng; Wang, Shurong; Liu, Xin; He, Yuxi; Li, Ying; Zhang, Yan

    2016-04-01

    The human cornea, consisting of five layers, is the transparent tissue that refracts and transmits light to the lens and retina, providing about two thirds of the refractive power of the eye. The stroma layer comprises nearly 90 % of the thickness of the cornea and thus plays a pivotal role in normal visual function. The bulk of this layer is constituted by proteins in the extracellular martrix secreted by the corneal epithelial, stroma, and endothelial cells. Clinical research has shown that corneal stroma diseases are common and involve conditions such as infections, injuries, and genetic defects, which cause severe visual disturbances or even blindness. To improve our understanding of the basic molecular mechanisms involved in the physiological and pathological activities of the corneal stroma, its proteins have been brought into the limelight to determine their crucial and irreplaceable roles. The data presented in a previous study have demonstrated the presence of 1679 proteins in the stroma, and this data set has subsequently been perfected by utilizing a highly sensitive isobaric peptide-labeling approach. According to their manifestations, these proteins can be classified as a gel-like organic material composed of proteoglycans, enzymes, and hemocyanin-binding proteins and a network of filaments composed of collagen, elastin, keratin, vimentin, and interconnected filaments comprising fibronectin and laminin. The aim of this review is to describe some corneal stroma proteins by highlighting their major functions and valuable applications in ophthalmologic research toward the better characterization and treatment of eye diseases.

  17. Human milk proteins: an interactomics and updated functional overview.

    PubMed

    D'Alessandro, Angelo; Scaloni, Andrea; Zolla, Lello

    2010-07-02

    Milk and milk fractions are characterized by a wide array of proteins, whose concentration spans across several orders of magnitude. By exploiting a combined approach based on functional gene ontology enrichment (FatiGO/Babelomics), hierarchical clustering, and pathway and network analyses, we merged data from literature dealing with protein-oriented studies on human milk. A total of 285 entries defined a nonredundant list upon comparison with the Ingenuity Knowledge Base from the Ingenuity Pathway Analysis software. Results were compared with an inventory of bovine milk proteins gathered from dedicated proteomic studies. A protein core of 106 proteins was found, with most of the entries associated to three main biological functions, namely nutrient transport/lipid metabolism, concretization of the immune system response and cellular proliferation processes. Our analyses confirm and emphasize that the biological role of the human milk proteins is not only limited to the provision of external nutrients and defense molecules against pathogens to the suckling but also to the direct stimulation of the growth of neonate tissues/organs and to the development of a proper independent immune system, both through the induction of a number of molecular cascades associated with cell proliferation/differentiation. The latter aspects were previously investigated by single-molecule dedicated studies, missing the holistic view that results from our analysis.

  18. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

  19. Lipid transfer proteins: classification, nomenclature, structure, and function.

    PubMed

    Salminen, Tiina A; Blomqvist, Kristina; Edqvist, Johan

    2016-11-01

    The non-specific lipid transfer proteins (LTPs) constitute a large protein family found in all land plants. They are small proteins characterized by a tunnel-like hydrophobic cavity, which makes them suitable for binding and transporting various lipids. The LTPs are abundantly expressed in most tissues. In general, they are synthesized with an N-terminal signal peptide that localizes the protein to spaces exterior to the plasma membrane. The in vivo functions of LTPs are still disputed, although evidence has accumulated for a role in the synthesis of lipid barrier polymers, such as cuticular waxes, suberin, and sporopollenin. There are also reports suggesting that LTPs are involved in signaling during pathogen attacks. LTPs are considered as key proteins for the plant's survival and colonization of land. In this review, we aim to present an overview of the current status of LTP research and also to discuss potential future applications of these proteins. We update the knowledge on 3D structures and lipid binding and review the most recent data from functional investigations, such as from knockout or overexpressing experiments. We also propose and argument for a novel system for the classification and naming of the LTPs.

  20. Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

    PubMed Central

    Gsponer, Jörg; Babu, M. Madan

    2012-01-01

    Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  1. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    PubMed Central

    Wu, Nicholas C.; Olson, C. Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q.; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-01-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available. PMID:26132554

  2. Quantitative assessment of protein function prediction from metagenomics shotgun sequences.

    PubMed

    Harrington, E D; Singh, A H; Doerks, T; Letunic, I; von Mering, C; Jensen, L J; Raes, J; Bork, P

    2007-08-28

    To assess the potential of protein function prediction in environmental genomics data, we analyzed shotgun sequences from four diverse and complex habitats. Using homology searches as well as customized gene neighborhood methods that incorporate intergenic and evolutionary distances, we inferred specific functions for 76% of the 1.4 million predicted ORFs in these samples (83% when nonspecific functions are considered). Surprisingly, these fractions are only slightly smaller than the corresponding ones in completely sequenced genomes (83% and 86%, respectively, by using the same methodology) and considerably higher than previously thought. For as many as 75,448 ORFs (5% of the total), only neighborhood methods can assign functions, illustrated here by a previously undescribed gene associated with the well characterized heme biosynthesis operon and a potential transcription factor that might regulate a coupling between fatty acid biosynthesis and degradation. Our results further suggest that, although functions can be inferred for most proteins on earth, many functions remain to be discovered in numerous small, rare protein families.

  3. Controlling multi-function of biomaterials interfaces based on multiple and competing adsorption of functional proteins.

    PubMed

    Guan, Zhen-Yu; Huang, Chao-Wei; Huang, Mei-Ching; Wu, Chih-Yu; Liu, Hui-Yu; Ding, Shih-Torng; Chen, Hsien-Yeh

    2017-01-01

    Multifunctional biomaterial surfaces can be created by controlling the competing adsorption of multiple proteins. To demonstrate this concept, bone morphogenetic protein 2 (BMP-2) and fibronectin were adsorbed to the hydrophobic surface of polychloro-para-xylylene. The resulting adsorption properties on the surface depended on the dimensional and steric characteristics of the selected protein molecule, the degree of denaturation of the adsorbed proteins, the associated adsorption of interphase water molecules within the protein layers, and the aggregation of proteins in a planar direction with respect to the adsorbent surface. Additionally, a defined surface composition was formed by the competing adsorption of multiple proteins, and this surface composition was directly linked to the composition of the protein mixture in the solution phase. Although the mechanism of this complex competing adsorption process is not fully understood, the adsorbed proteins were irreversibly adsorbed and were unaffected by the further adsorption of homologous or heterologous proteins. Moreover, synergistic biological activities, including cell osteogenesis and proliferation independently and specifically induced by BMP-2 or fibronectin, were observed on the modified surface, and these biological activities were positively correlated with the surface composition of the multiple adsorbed proteins. These results provide insights and important design parameters for prospective biomaterials and biointerfaces for (multi)functional modifications. The ability to control protein/interface properties will be beneficial for the processing of biomaterials for clinical applications and industrial products.

  4. Emerging functions of the unfolded protein response in immunity

    PubMed Central

    Janssens, Sophie; Pulendran, Bali; Lambrecht, Bart N.

    2015-01-01

    The unfolded protein response (UPR) has traditionally been viewed as an adaptive response triggered upon accumulation of unfolded proteins in the endoplasmic reticulum (ER), aimed at restoring ER function. The UPR can also be an anticipatory response that is activated well before the disruption of protein homeostasis. UPR signaling intersects at many levels with the innate and adaptive immune response. In some immune cell types like dendritic cells and B cells, particular UPR sensors appear constitutively active in the absence of traditional UPR gene program induction, necessary for antigen presentation and immunoglobulin synthesis. The UPR also influences Toll-like receptor signaling and NF-κB activation, and some pathogens subvert the UPR. This review summarizes these emerging non-canonical functions of the UPR in immunity. PMID:25232821

  5. Comprehensive functional analysis of large lists of genes and proteins.

    PubMed

    Mlecnik, Bernhard; Galon, Jérôme; Bindea, Gabriela

    2017-03-22

    The interpretation of high dimensional datasets resulting from genomic and proteomic experiments in a timely and efficient manner is challenging. ClueGO software is a Cytoscape App that extracts representative functional biological information for large lists of genes or proteins. The functional enrichment analysis is based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through ClueGO. Predefined settings for the selection of the terms are provided to facilitate the analysis. Results are visualized as networks in which Gene Ontology (GO) terms and pathways are grouped based on their biological role. Many species are now supported by ClueGO and additional organisms are added on demand. ClueGO can be used together with the CluePedia App to enable the visualization of protein-protein interactions within or between pathways.

  6. Functionality of Gliadin Proteins in Wheat Flour Tortillas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gliadins are monomeric proteins that are encoded by the genes at the locus Gli 1 and Gli 2 present on the short arm of homeologous wheat chromosomes 1 and 6, respectively. Studies have suggested that gliadins may play an important role in determining the functional properties of wheat flour. The mai...

  7. CBS domains: structure, function, and pathology in human proteins.

    PubMed

    Ignoul, Sofie; Eggermont, Jan

    2005-12-01

    The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

  8. Fundamental Characteristics of AAA+ Protein Family Structure and Function

    PubMed Central

    2016-01-01

    Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

  9. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  10. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting.

    PubMed

    Rothman, J H; Raymond, C K; Gilbert, T; O'Hara, P J; Stevens, T H

    1990-06-15

    Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.

  11. Domain mobility in proteins: functional and evolutionary implications.

    PubMed

    Basu, Malay Kumar; Poliakov, Eugenia; Rogozin, Igor B

    2009-05-01

    A substantial fraction of eukaryotic proteins contains multiple domains, some of which show a tendency to occur in diverse domain architectures and can be considered mobile (or 'promiscuous'). These promiscuous domains are typically involved in protein-protein interactions and play crucial roles in interaction networks, particularly those contributing to signal transduction. They also play a major role in creating diversity of protein domain architecture in the proteome. It is now apparent that promiscuity is a volatile and relatively fast-changing feature in evolution, and that only a few domains retain their promiscuity status throughout evolution. Many such domains attained their promiscuity status independently in different lineages. Only recently, we have begun to understand the diversity of protein domain architectures and the role the promiscuous domains play in evolution of this diversity. However, many of the biological mechanisms of protein domain mobility remain shrouded in mystery. In this review, we discuss our present understanding of protein domain promiscuity, its evolution and its role in cellular function.

  12. Epicutaneous exposure to proteins and skin immune function.

    PubMed

    Kimber, Ian; Griffiths, Christopher E M; Basketter, David A; McFadden, John P; Dearman, Rebecca J

    2014-01-01

    The skin has a sophisticated and highly orchestrated immune system. The ability of proteins encountered at skin surfaces to access that immune system remains controversial, however. In this article the question considered is whether proteins encountered epicutaneously (on the skin) at abraded or tape-stripped skin surfaces, but also at sites where the skin is intact, can engage with the cutaneous immune system to provoke and regulate responses. The available evidence suggests that epicutaneous exposure to foreign proteins is able to elicit immune and allergic responses, and that encounter with protein via this route may favour the development of selective Th2 responses and allergic sensitisation. It is also clear that proteins can modify immunological function when delivered topically and that intact skin may provide an effective route of exposure for active immunotherapy of allergic disease. An appreciation that epicutaneously applied proteins can interact with the skin immune system, even when delivered at intact skin sites, opens up important opportunities for immunotherapy, local immune modulation and the treatment of inflammatory skin diseases. It also indicates that this route of exposure must be considered as part of the safety assessment and risk management of protein-induced allergic sensitisation.

  13. Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

    PubMed

    Brioschi, Maura; Polvani, Gianluca; Fratto, Pasquale; Parolari, Alessandro; Agostoni, Piergiuseppe; Tremoli, Elena; Banfi, Cristina

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.

  14. Redox Proteomics Identification of Oxidatively Modified Myocardial Proteins in Human Heart Failure: Implications for Protein Function

    PubMed Central

    Brioschi, Maura; Polvani, Gianluca; Fratto, Pasquale; Parolari, Alessandro; Agostoni, Piergiuseppe; Tremoli, Elena; Banfi, Cristina

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF. PMID:22606238

  15. A three-way approach for protein function classification

    PubMed Central

    2017-01-01

    The knowledge of protein functions plays an essential role in understanding biological cells and has a significant impact on human life in areas such as personalized medicine, better crops and improved therapeutic interventions. Due to expense and inherent difficulty of biological experiments, intelligent methods are generally relied upon for automatic assignment of functions to proteins. The technological advancements in the field of biology are improving our understanding of biological processes and are regularly resulting in new features and characteristics that better describe the role of proteins. It is inevitable to neglect and overlook these anticipated features in designing more effective classification techniques. A key issue in this context, that is not being sufficiently addressed, is how to build effective classification models and approaches for protein function prediction by incorporating and taking advantage from the ever evolving biological information. In this article, we propose a three-way decision making approach which provides provisions for seeking and incorporating future information. We considered probabilistic rough sets based models such as Game-Theoretic Rough Sets (GTRS) and Information-Theoretic Rough Sets (ITRS) for inducing three-way decisions. An architecture of protein functions classification with probabilistic rough sets based three-way decisions is proposed and explained. Experiments are carried out on Saccharomyces cerevisiae species dataset obtained from Uniprot database with the corresponding functional classes extracted from the Gene Ontology (GO) database. The results indicate that as the level of biological information increases, the number of deferred cases are reduced while maintaining similar level of accuracy. PMID:28234929

  16. cncRNAs: Bi-functional RNAs with protein coding and non-coding functions.

    PubMed

    Kumari, Pooja; Sampath, Karuna

    2015-12-01

    For many decades, the major function of mRNA was thought to be to provide protein-coding information embedded in the genome. The advent of high-throughput sequencing has led to the discovery of pervasive transcription of eukaryotic genomes and opened the world of RNA-mediated gene regulation. Many regulatory RNAs have been found to be incapable of protein coding and are hence termed as non-coding RNAs (ncRNAs). However, studies in recent years have shown that several previously annotated non-coding RNAs have the potential to encode proteins, and conversely, some coding RNAs have regulatory functions independent of the protein they encode. Such bi-functional RNAs, with both protein coding and non-coding functions, which we term as 'cncRNAs', have emerged as new players in cellular systems. Here, we describe the functions of some cncRNAs identified from bacteria to humans. Because the functions of many RNAs across genomes remains unclear, we propose that RNAs be classified as coding, non-coding or both only after careful analysis of their functions.

  17. Optimization of functionalization conditions for protein analysis by AFM

    NASA Astrophysics Data System (ADS)

    Arroyo-Hernández, María; Daza, Rafael; Pérez-Rigueiro, Jose; Elices, Manuel; Nieto-Márquez, Jorge; Guinea, Gustavo V.

    2014-10-01

    Activated vapor silanization (AVS) is used to functionalize silicon surfaces through deposition of amine-containing thin films. AVS combines vapor silanization and chemical vapor deposition techniques and allows the properties of the functionalized layers (thickness, amine concentration and topography) to be controlled by tuning the deposition conditions. An accurate characterization is performed to correlate the deposition conditions and functional-film properties. In particular, it is shown that smooth surfaces with a sufficient surface density of amine groups may be obtained with this technique. These surfaces are suitable for the study of proteins with atomic force microscopy.

  18. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  19. Modulation of Sertoli cell secretory function by rat round spermatid protein(s).

    PubMed

    Onoda, M; Djakiew, D

    1990-10-01

    The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 micrograms/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 micrograms/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 micrograms/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 micrograms/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

  20. Influence of processing on functionality of milk and dairy proteins.

    PubMed

    Augustin, Mary Ann; Udabage, Punsandani

    2007-01-01

    The inherent physical functionality of dairy ingredients makes them useful in a range of food applications. These functionalities include their solubility, water binding, viscosity, gelation, heat stability, renneting, foaming, and emulsifying properties. The suitability of dairy ingredients for an application can be further tailored by altering the structure of the proteins using appropriate processes. The processes discussed include physical modification (heat treatment, acidification, addition of mineral slats, homogenization, and shear), enzymatic modification (renneting, hydrolysis, and transglutamination), and chemical modification (use of chemical agents and the Maillard reaction). Emerging food processes (high pressure and ultrasound) are also discussed. The challenges for using dairy ingredients for the delivery of nutrients and bioactive components, while maintaining physical functionality, are also highlighted. There is a need for continued research into the fundamental aspects of milk proteins and their responses to various stresses for further differentiation of milk products and for the delivery of ingredients with consistent quality for target applications.

  1. Senescence Marker Protein 30: Functional and Structural Insights to its Unknown Physiological Function

    PubMed Central

    Scott, Stephanie H.; Bahnson, Brian J.

    2011-01-01

    Senescence marker protein 30 (SMP30) is a multifunctional protein involved in cellular Ca2+ homeostasis and the biosynthesis of ascorbate in non-primate mammals. The primary structure of the protein is highly conserved among vertebrates, suggesting the existence of a significant physiological function common to all mammals, including primates. Enzymatic activities of SMP30 include aldonolactone and organophosphate hydrolysis. Protective effects against apoptosis and oxidative stress have been reported. X-ray crystallography revealed that SMP30 is a six-bladed β-propeller with structural similarity to paraoxonase 1, another protein with lactonase and organophosphate hydrolase activities. SMP30 has recently been tied to several physiological conditions including osteoporosis, liver fibrosis, diabetes, and cancer. This review aims to describe the recent advances made toward understanding the connection between molecular structure, enzymatic activity and physiological function of this highly conserved, multifaceted protein. PMID:22844387

  2. Expressed Protein Ligation: A Resourceful Tool to Study Protein Structure and Function

    PubMed Central

    Berrade, Luis; Camarero, Julio A.

    2013-01-01

    This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function. PMID:19685006

  3. Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

    PubMed Central

    VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

    2011-01-01

    Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

  4. Computational design of receptor and sensor proteins with novel functions

    NASA Astrophysics Data System (ADS)

    Looger, Loren L.; Dwyer, Mary A.; Smith, James J.; Hellinga, Homme W.

    2003-05-01

    The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, L-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or L-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.

  5. [Histidine triad protein superfamily--biological function and enzymatic activity].

    PubMed

    Krakowiak, Agnieszka; Fryc, Izabela

    2012-01-01

    The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

  6. Muscarinic receptor family interacting proteins: role in receptor function.

    PubMed

    Borroto-Escuela, Dasiel O; Correia, Patrícia A; Romero-Fernandez, Wilber; Narvaez, Manuel; Fuxe, Kjell; Ciruela, Francisco; Garriga, Pere

    2011-02-15

    G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

  7. Prediction of functional residues in water channels and related proteins.

    PubMed Central

    Froger, A.; Tallur, B.; Thomas, D.; Delamarche, C.

    1998-01-01

    In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico-chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport. PMID:9655351

  8. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

    PubMed

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application.

  9. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry

    PubMed Central

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application. PMID:26761849

  10. The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*

    PubMed Central

    Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin

    2013-01-01

    The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization. PMID:23553632

  11. Eliciting the Functional Taxonomy from protein annotations and taxa

    PubMed Central

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-01-01

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules. PMID:27534507

  12. Eliciting the Functional Taxonomy from protein annotations and taxa.

    PubMed

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-08-18

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules.

  13. Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila

    PubMed Central

    Gaertig, Jacek; Wloga, Dorota; Vasudevan, Krishna Kumar; Guha, Mayukh; Dentler, William

    2015-01-01

    The ciliate Tetrahymena thermophila is an excellent model system for the discovery and functional studies of ciliary proteins. The power of the model is based on the ease with which cilia can be purified in large quantities for fractionation and proteomic identification, and the ability to knock out any gene by homologous DNA recombination. Here, we include methods used by our laboratories for isolation and fractionation of cilia, in vivo tagging and localization of ciliary proteins and the evaluation of ciliary mutants. PMID:23522474

  14. Membrane Proteins in Four Acts: Function Precedes Structure Determination

    PubMed Central

    Cramer, W. A.; Zakharov, S. D.; Hasan, S. Saif; Zhang, H.; Baniulis, D.; Zhalnina, M. V.; Soriano, G. M.; Sharma, O.; Rochet, J. C.; Ryan, C.; Whitelegge., J.; Kurisu, G.; Yamashita, E.

    2011-01-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/ proton translocation). (1) Crystal structures of the eight subunit heterooligomeric trans-membrane dimeric cytochrome b6f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of seventeen monotopic and polytopic hetero-subunits. (II) β-barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins (1). A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained (2). A crystal structure of the N-terminal translocation domain of colicin E3

  15. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  16. Prebiotic Alternatives to Proteins: Structure and Function of Hyperbranched Polyesters

    NASA Astrophysics Data System (ADS)

    Mamajanov, Irena; Callahan, Michael P.; Dworkin, Jason P.; Cody, George D.

    2015-06-01

    Proteins are responsible multiple biological functions, such as ligand binding, catalysis, and ion channeling. This functionality is enabled by proteins' three-dimensional structures that require long polypeptides. Since plausibly prebiotic synthesis of functional polypeptides has proven challenging in the laboratory, we propose that these functions may have been initially performed by alternative macromolecular constructs, namely hyperbranched polymers (HBPs), during early stages of chemical evolution. HBPs can be straightforwardly synthesized in one-pot processes, possess globular structures determined by their architecture as opposed to folding in proteins, and have documented ligand binding and catalytic properties. Our initial study focuses on glycerol-citric acid HBPs synthesized via moderate heating in the dry state. The polymerization products consisted of a mixture of isomeric structures of varying molar mass as evidenced by NMR, mass spectrometry and size-exclusion chromatography. Addition of divalent cations during polymerization resulted in increased incorporation of citric acid into the HBPs and the possible formation of cation-oligomer complexes. The chelating properties of citric acid govern the makeup of the resulting polymer, turning the polymerization system into a rudimentary smart material.

  17. Functions of BET proteins in erythroid gene expression.

    PubMed

    Stonestrom, Aaron J; Hsu, Sarah C; Jahn, Kristen S; Huang, Peng; Keller, Cheryl A; Giardine, Belinda M; Kadauke, Stephan; Campbell, Amy E; Evans, Perry; Hardison, Ross C; Blobel, Gerd A

    2015-04-30

    Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

  18. Protein function prediction using local 3D templates.

    PubMed

    Laskowski, Roman A; Watson, James D; Thornton, Janet M

    2005-08-19

    The prediction of a protein's function from its 3D structure is becoming more and more important as the worldwide structural genomics initiatives gather pace and continue to solve 3D structures, many of which are of proteins of unknown function. Here, we present a methodology for predicting function from structure that shows great promise. It is based on 3D templates that are defined as specific 3D conformations of small numbers of residues. We use four types of template, covering enzyme active sites, ligand-binding residues, DNA-binding residues and reverse templates. The latter are templates generated from the target structure itself and scanned against a representative subset of all known protein structures. Together, the templates provide a fairly thorough coverage of the known structures and ensure that if there is a match to a known structure it is unlikely to be missed. A new scoring scheme provides a highly sensitive means of discriminating between true positive and false positive template matches. In all, the methodology provides a powerful new tool for function prediction to complement those already in use.

  19. TMEM16 proteins: unknown structure and confusing functions

    PubMed Central

    Picollo, Alessandra; Malvezzi, Mattia; Accardi, Alessio

    2014-01-01

    The TMEM16 family of membrane proteins, also known as anoctamins, play key roles in a variety of physiological functions that range from ion transport, to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca2+-activated Cl− channels (CaCCs) and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The role(s) of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7), and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transport. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated. PMID:25451786

  20. Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center

    SciTech Connect

    Kim, Sung-Hou; Shin, Dong Hae; Hou, Jingtong; Chandonia, John-Marc; Das, Debanu; Choi, In-Geol; Kim, Rosalind; Kim, Sung-Hou

    2007-09-02

    Advances in sequence genomics have resulted in an accumulation of a huge number of protein sequences derived from genome sequences. However, the functions of a large portion of them cannot be inferred based on the current methods of sequence homology detection to proteins of known functions. Three-dimensional structure can have an important impact in providing inference of molecular function (physical and chemical function) of a protein of unknown function. Structural genomics centers worldwide have been determining many 3-D structures of the proteins of unknown functions, and possible molecular functions of them have been inferred based on their structures. Combined with bioinformatics and enzymatic assay tools, the successful acceleration of the process of protein structure determination through high throughput pipelines enables the rapid functional annotation of a large fraction of hypothetical proteins. We present a brief summary of the process we used at the Berkeley Structural Genomics Center to infer molecular functions of proteins of unknown function.

  1. Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center.

    PubMed

    Shin, Dong Hae; Hou, Jingtong; Chandonia, John-Marc; Das, Debanu; Choi, In-Geol; Kim, Rosalind; Kim, Sung-Hou

    2007-09-01

    Advances in sequence genomics have resulted in an accumulation of a huge number of protein sequences derived from genome sequences. However, the functions of a large portion of them cannot be inferred based on the current methods of sequence homology detection to proteins of known functions. Three-dimensional structure can have an important impact in providing inference of molecular function (physical and chemical function) of a protein of unknown function. Structural genomics centers worldwide have been determining many 3-D structures of the proteins of unknown functions, and possible molecular functions of them have been inferred based on their structures. Combined with bioinformatics and enzymatic assay tools, the successful acceleration of the process of protein structure determination through high throughput pipelines enables the rapid functional annotation of a large fraction of hypothetical proteins. We present a brief summary of the process we used at the Berkeley Structural Genomics Center to infer molecular functions of proteins of unknown function.

  2. Protein function in precision medicine: deep understanding with machine learning.

    PubMed

    Rost, Burkhard; Radivojac, Predrag; Bromberg, Yana

    2016-08-01

    Precision medicine and personalized health efforts propose leveraging complex molecular, medical and family history, along with other types of personal data toward better life. We argue that this ambitious objective will require advanced and specialized machine learning solutions. Simply skimming some low-hanging results off the data wealth might have limited potential. Instead, we need to better understand all parts of the system to define medically relevant causes and effects: how do particular sequence variants affect particular proteins and pathways? How do these effects, in turn, cause the health or disease-related phenotype? Toward this end, deeper understanding will not simply diffuse from deeper machine learning, but from more explicit focus on understanding protein function, context-specific protein interaction networks, and impact of variation on both.

  3. A genetic system to study Plasmodium falciparum protein function.

    PubMed

    Birnbaum, Jakob; Flemming, Sven; Reichard, Nick; Soares, Alexandra Blancke; Mesén-Ramírez, Paolo; Jonscher, Ernst; Bergmann, Bärbel; Spielmann, Tobias

    2017-03-13

    Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

  4. Defining the boundaries: structure and function of LOB domain proteins.

    PubMed

    Majer, Christine; Hochholdinger, Frank

    2011-01-01

    The plant-specific LBD (Lateral Organ Boundaries Domain) gene family is essential in the regulation of plant lateral organ development and is involved in the regulation of anthocyanin and nitrogen metabolism. LBD proteins contain a characteristic LOB domain composed of a C-motif required for DNA-binding, a conserved glycine residue, and a leucine-zipper-like sequence required for protein-protein interactions. Recently, several LBD genes associated with mutant phenotypes related to almost all aspects of plant development, including embryo, root, leaf, and inflorescence development have been functionally characterized. These novel insights contribute to a better understanding of the molecular definition of boundaries between organs or boundaries between organs and meristems and the regulation of these processes by environmental cues and phytohormones.

  5. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1^E4, these kinases regulate one of

  6. Finding sequence motifs in groups of functionally related proteins.

    PubMed

    Smith, H O; Annau, T M; Chandrasegaran, S

    1990-01-01

    We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.

  7. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

    PubMed Central

    Gillooly, D J; Simonsen, A; Stenmark, H

    2001-01-01

    PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling. PMID:11284710

  8. The evolution of function in strictosidine synthase-like proteins.

    PubMed

    Hicks, Michael A; Barber, Alan E; Giddings, Lesley-Ann; Caldwell, Jenna; O'Connor, Sarah E; Babbitt, Patricia C

    2011-11-01

    The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural, and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called strictosidine synthase-like (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins.

  9. Graphlet kernels for prediction of functional residues in protein structures.

    PubMed

    Vacic, Vladimir; Iakoucheva, Lilia M; Lonardi, Stefano; Radivojac, Predrag

    2010-01-01

    We introduce a novel graph-based kernel method for annotating functional residues in protein structures. A structure is first modeled as a protein contact graph, where nodes correspond to residues and edges connect spatially neighboring residues. Each vertex in the graph is then represented as a vector of counts of labeled non-isomorphic subgraphs (graphlets), centered on the vertex of interest. A similarity measure between two vertices is expressed as the inner product of their respective count vectors and is used in a supervised learning framework to classify protein residues. We evaluated our method on two function prediction problems: identification of catalytic residues in proteins, which is a well-studied problem suitable for benchmarking, and a much less explored problem of predicting phosphorylation sites in protein structures. The performance of the graphlet kernel approach was then compared against two alternative methods, a sequence-based predictor and our implementation of the FEATURE framework. On both tasks, the graphlet kernel performed favorably; however, the margin of difference was considerably higher on the problem of phosphorylation site prediction. While there is data that phosphorylation sites are preferentially positioned in intrinsically disordered regions, we provide evidence that for the sites that are located in structured regions, neither the surface accessibility alone nor the averaged measures calculated from the residue microenvironments utilized by FEATURE were sufficient to achieve high accuracy. The key benefit of the graphlet representation is its ability to capture neighborhood similarities in protein structures via enumerating the patterns of local connectivity in the corresponding labeled graphs.

  10. Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster

    PubMed Central

    Cassar, Marlène; Kretzschmar, Doris

    2016-01-01

    The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer’s Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation. PMID:27507933

  11. Characterization of an upstream regulatory element of adenovirus L1 poly (A) site.

    PubMed

    Liu, Li

    2005-06-20

    The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1-L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532-541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977-5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682-4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3' end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207-2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state m

  12. Critical importance of RAB proteins for synaptic function.

    PubMed

    Mignogna, Maria Lidia; D'Adamo, Patrizia

    2017-02-01

    Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity.

  13. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    NASA Astrophysics Data System (ADS)

    Millhauser, Glenn L.

    2007-05-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

  14. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2007-01-01

    The present invention provides a method for production of functional proteins including hormones by renal cells in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-a-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  15. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2004-01-01

    The present invention provides a method for production of functional proteins including hormones by renal cells in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-a-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  16. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2011-01-01

    A method for the production of functional proteins including hormones by renal cells in a three dimensional culturing process responsive to shear stress uses a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-.alpha.-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D.sub.3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating an in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  17. Functional properties of tropical banded cricket (Gryllodes sigillatus) protein hydrolysates.

    PubMed

    Hall, Felicia G; Jones, Owen G; O'Haire, Marguerite E; Liceaga, Andrea M

    2017-06-01

    Recently, the benefits of entomophagy have been widely discussed. Due to western cultures' reluctance, entomophagy practices are leaning more towards incorporating insects into food products. In this study, whole crickets (Gryllodes sigillatus) were hydrolyzed with alcalase at 0.5, 1.5, and 3.0% (w/w) for 30, 60, and 90min. Degree of hydrolysis (DH), amino acid composition, solubility, emulsion and foaming properties were evaluated. Hydrolysis produced peptides with 26-52% DH compared to the control containing no enzyme (5% DH). Protein solubility of hydrolysates improved (p<0.05) over a range of pH's, exhibiting >30% soluble protein at pH 3 and 7 and 50-90% at alkaline pH, compared with the control. Emulsion activity index ranged from 7 to 32m(2)/g, while foamability ranged from 100 to 155% for all hydrolysates. These improved functional properties demonstrate the potential to develop cricket protein hydrolysates as a source of functional alternative protein in food ingredient formulations.

  18. PROSNET: INTEGRATING HOMOLOGY WITH MOLECULAR NETWORKS FOR PROTEIN FUNCTION PREDICTION

    PubMed Central

    Wang, Sheng; Qu, Meng

    2016-01-01

    Automated annotation of protein function has become a critical task in the post-genomic era. Network-based approaches and homology-based approaches have been widely used and recently tested in large-scale community-wide assessment experiments. It is natural to integrate network data with homology information to further improve the predictive performance. However, integrating these two heterogeneous, high-dimensional and noisy datasets is non-trivial. In this work, we introduce a novel protein function prediction algorithm ProSNet. An integrated heterogeneous network is first built to include molecular networks of multiple species and link together homologous proteins across multiple species. Based on this integrated network, a dimensionality reduction algorithm is introduced to obtain compact low-dimensional vectors to encode proteins in the network. Finally, we develop machine learning classification algorithms that take the vectors as input and make predictions by transferring annotations both within each species and across different species. Extensive experiments on five major species demonstrate that our integration of homology with molecular networks substantially improves the predictive performance over existing approaches. PMID:27896959

  19. Functionalized periodic mesoporous organosilicas for selective adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Zhu, Ling; Liu, Xiaoyan; Chen, Tong; Xu, Zhigang; Yan, Wenfu; Zhang, Haixia

    2012-07-01

    The periodic mesoporous organosilicas (PMO) with an organobridged (sbnd CH2sbnd ) was synthesized and functionalized with amino or carboxylic groups by post-synthesis methods. The functionalized PMO by changing the hydrophilic/hydrophobic property and the net charge could be used to selectively adsorb and purify proteins with different shapes and different isoelectric points (pI). The experimental result showed that Bovine serum albumin (BSA) was adsorbed quicker than hemoglobin (Hb) on the materials, and lysozyme (Lys) could not be adsorbed on these PMO materials at all. The adsorption capacity of amino groups modified PMO (PMO-(NH2)2) for BSA was 44.67 mg/g and 300.0 mg/gfor Hb on carboxylic groups modified PMO (PMO-(COOH)2). The adsorption behavior of proteins was affected strongly by the interaction among different constituents in the mixture of proteins. In addition, it is found that the adsorption rate of (PMO-(NH2)2 for adsorption of proteins was much slower than PMO-(COOH)2.

  20. Structural and functional characterization of synapse-associated protein-97

    NASA Astrophysics Data System (ADS)

    Wang, Lei

    Synapse-associated protein-97 (SAP97) as a scaffold protein plays an important role in regulating neural signal transmission in the central nervous system by coupling with activated membrane receptors, ion channels, and downstream signaling proteins. SAP97 consists of six functional domains: L27, PDZ1, PDZ2, PDZ3, SH3, and GK. Each of these domains mediates the interactions of SAP97 with other proteins. Understanding the molecular mechanism of these interactions in neural signal transmission is a goal of this study. Here high-resolution nuclear magnetic resonance spectroscopy and fluorescence anisotropy are employed towards the goal of the structural and functional characterization of SAP97; specifically, we (a) characterize the binding of the PDZ domains of SAP97 with the C-terminus of NR2B, and determine the structure of the PDZ1-NR2B; (b) characterize the binding of the PDZ domains with the C-terminus of stargazin and multiple mutants, and identify the perturbed amino acids in PDZ2 upon the binding of stargazin; (c) characterize the binding specificity carried by the beta2/beta3 loop of the PDZ3 domain. These results provide insight into the molecular mechanism for the binding specificities of the PDZ domains of SAP97, thereby furthering the development of drugs that target these domains to treat neurological diseases.

  1. Functional and technological properties of camel milk proteins: a review.

    PubMed

    Hailu, Yonas; Hansen, Egon Bech; Seifu, Eyassu; Eshetu, Mitiku; Ipsen, Richard; Kappeler, Stefan

    2016-11-01

    This review summarises current knowledge on camel milk proteins, with focus on significant peculiarities in protein composition and molecular properties. Camel milk is traditionally consumed as a fresh or naturally fermented product. Within the last couple of years, an increasing quantity is being processed in dairy plants, and a number of consumer products have been marketed. A better understanding of the technological and functional properties, as required for product improvement, has been gained in the past years. Absence of the whey protein β-LG and a low proportion of к-casein cause differences in relation to dairy processing. In addition to the technological properties, there are also implications for human nutrition and camel milk proteins are of interest for applications in infant foods, for food preservation and in functional foods. Proposed health benefits include inhibition of the angiotensin converting enzyme, antimicrobial and antioxidant properties as well as an antidiabetogenic effect. Detailed investigations on foaming, gelation and solubility as well as technological consequences of processing should be investigated further for the improvement of camel milk utilisation in the near future.

  2. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    PubMed

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  3. Interaction networks: from protein functions to drug discovery. A review.

    PubMed

    Chautard, E; Thierry-Mieg, N; Ricard-Blum, S

    2009-06-01

    Most genes, proteins and other components carry out their functions within a complex network of interactions and a single molecule can affect a wide range of other cell components. A global, integrative, approach has been developed for several years, including protein-protein interaction networks (interactomes). In this review, we describe the high-throughput methods used to identify new interactions and to build large interaction datasets. The minimum information required for reporting a molecular interaction experiment (MIMIx) has been defined as a standard for storing data in publicly available interaction databases. Several examples of interaction networks from molecular machines (proteasome) or organelles (phagosome, mitochondrion) to whole organisms (viruses, bacteria, yeast, fly, and worm) are given and attempts to cover the entire human interaction network are discussed. The methods used to perform the topological analysis of interaction networks and to extract biological information from them are presented. These investigations have provided clues on protein functions, signalling and metabolic pathways, and physiological processes, unraveled the molecular basis of some diseases (cancer, infectious diseases), and will be very useful to identify new therapeutic targets and for drug discovery. A major challenge is now to integrate data from different sources (interactome, transcriptome, phenome, localization) to switch from static to dynamic interaction networks. The merging of a viral interactome and the human interactome has been used to simulate viral infection, paving the way for future studies aiming at providing molecular basis of human diseases.

  4. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  5. Renal function, protein binding and pharmacological response to diazoxide.

    PubMed Central

    Pearson, R M; Breckenridge, A M

    1976-01-01

    1 The effect of rapid (10s) injections of diazoxide was studied in ten hypertensive patients with varying degrees of impairment of renal function. 2 There was a significant correlation between the patient's plasma urea concentration and reduction in mean arterial blood pressure. Diazoxide was also shown to be less highly protein bound in patients with renal failure. 3 It is suggested that the explanation for the increased hypotensive effect of diazoxide observed in patients with reduced renal function is related to higher unbound drug concentrations. PMID:973937

  6. Carotenoid Antenna Binding and Function in Retinal Proteins

    DTIC Science & Technology

    2012-08-13

    REPORT Carotenoid antenna binding and function in retinal proteins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Xanthorhodopsin, a proton pump from the...eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light... carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4

  7. Spatio-temporal coordination among functional residues in protein

    PubMed Central

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J.

    2017-01-01

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis. PMID:28091537

  8. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  9. Spatio-temporal coordination among functional residues in protein

    NASA Astrophysics Data System (ADS)

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J.

    2017-01-01

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis.

  10. Spatio-temporal coordination among functional residues in protein.

    PubMed

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J

    2017-01-16

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis.

  11. High Precision Prediction of Functional Sites in Protein Structures

    PubMed Central

    Buturovic, Ljubomir; Wong, Mike; Tang, Grace W.; Altman, Russ B.; Petkovic, Dragutin

    2014-01-01

    We address the problem of assigning biological function to solved protein structures. Computational tools play a critical role in identifying potential active sites and informing screening decisions for further lab analysis. A critical parameter in the practical application of computational methods is the precision, or positive predictive value. Precision measures the level of confidence the user should have in a particular computed functional assignment. Low precision annotations lead to futile laboratory investigations and waste scarce research resources. In this paper we describe an advanced version of the protein function annotation system FEATURE, which achieved 99% precision and average recall of 95% across 20 representative functional sites. The system uses a Support Vector Machine classifier operating on the microenvironment of physicochemical features around an amino acid. We also compared performance of our method with state-of-the-art sequence-level annotator Pfam in terms of precision, recall and localization. To our knowledge, no other functional site annotator has been rigorously evaluated against these key criteria. The software and predictive models are incorporated into the WebFEATURE service at http://feature.stanford.edu/wf4.0-beta. PMID:24632601

  12. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

    PubMed Central

    Alves, Murilo S.; Dadalto, Silvana P.; Gonçalves, Amanda B.; de Souza, Gilza B.; Barros, Vanessa A.; Fietto, Luciano G.

    2014-01-01

    Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP), amino-acid sequence WRKYGQK (WRKY), myelocytomatosis related proteins (MYC), myeloblastosis related proteins (MYB), APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP) and no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC) (NAC). We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses. PMID:28250372

  13. Control of protein adsorption on functionalized electrospun fibers.

    PubMed

    Grafahrend, Dirk; Calvet, Julia Lleixa; Klinkhammer, Kristina; Salber, Jochen; Dalton, Paul D; Möller, Martin; Klee, Doris

    2008-10-15

    Electrospun fibers that are protein resistant and functionalized with bioactive signals were produced by solution electrospinning amphiphilic block copolymers. Poly (ethylene glycol)-block-poly(D,L-lactide) (PEG-b-PDLLA) was synthesized in two steps, with a PEG segment of 10 kDa, while the PDLLA block ranged from 20 to 60 kDa. Depending on the PEG and PDLLA segment ratio, as well as solvent selection, the hydrophilicity and protein adsorption could be altered on the electrospun mesh. Furthermore, an alpha-acetal PEG-b-PDLLA was synthesized that allowed the conjugation of active molecules, resulting in surface functionalization of the electrospun fiber. Electrospun material with varying morphologies and diameter were electrospun from 10, 20, and 30 wt.% solutions. Sessile drop measurements showed a reduction in the contact angle from 120 degrees for pure poly(D,L-lactide) with increasing PEG/PDLLA ratio. All electrospun block PEG-b-PDLLA fibers had hydrophilic properties, with contact angles below 45 degrees . The fibers were collected onto six-arm star-poly(ethylene glycol) (star-PEG) coated silicon wafers and incubated with fluorescently labeled proteins. All PEG-b-PDLLA fibers showed no detectable adsorption of bovine serum albumin (BSA) independent of their composition while a dependence between hydrophobic block length was observed for streptavidin adsorption. Fibers of block copolymers with PDLLA blocks smaller than 39 kDa showed no adsorption of BSA or streptavidin, indicating good non-fouling properties. Fibers were surface functionalized with N(epsilon)-(+)-biotinyl-L-lysine (biocytin) or RGD peptide by attaching the molecule to the PEG block during synthesis. Protein adsorption measurements, and the controlled interaction of biocytin with fluorescently labeled streptavidin, showed that the electrospun fibers were both resistant to protein adsorption and are functionalized. Fibroblast adhesion was contrasting between the unfunctionalized and RGD

  14. DND protein functions as a translation repressor during zebrafish embryogenesis.

    PubMed

    Kobayashi, Manami; Tani-Matsuhana, Saori; Ohkawa, Yasuka; Sakamoto, Hiroshi; Inoue, Kunio

    2017-03-04

    Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish.

  15. 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.

  16. Maximizing the functional lifetime of protein a resins.

    PubMed

    Zhang, Jennifer; Siva, Sethu; Caple, Ryan; Ghose, Sanchayita; Gronke, Rob

    2017-02-20

    Protein A chromatography is currently the industry gold-standard for monoclonal antibody and Fc-fusion protein purification. The high cost of Protein A, however, makes resin lifetime and resin reuse an important factor for process economics. Typical resin lifetime studies performed in the industry usually examine the effect of resin re-use on binding capacity, yield, and product quality without answering the fundamental question of what is causing the decrease in performance. A two part mechanistic study was conducted in an attempt to decouple the effect of the two possible factors (resin hydrolysis and/or degradation vs. resin fouling) on column performance over lifetime of the most commonly used alkali-stable Protein A resins (MabSelect SuRe and MabSelect SuRe LX). The change in binding capacity as a function of sodium hydroxide concentration (rate of hydrolysis), temperature, and stabilizing additives was examined. Additionally, resin extraction studies and product cycling studies were conducted to determine cleaning effectiveness (resin fouling) of various cleaning strategies. Sodium hydroxide-based cleaning solutions were shown to be more effective at preventing resin fouling. Conversely, cold temperature and the use of stabilizing additives in conjunction with sodium hydroxide were found to be beneficial in minimizing the rate of Protein A ligand hydrolysis. An effective and robust cleaning strategy is presented here to maximize resin lifetime and thereby the number of column cycles for future manufacturing processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017.

  17. Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics.

    PubMed

    Tiwari, Sadhana; Vinchurkar, Madhuri; Rao, V Ramgopal; Garnier, Gil

    2017-03-02

    Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept.

  18. Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics

    NASA Astrophysics Data System (ADS)

    Tiwari, Sadhana; Vinchurkar, Madhuri; Rao, V. Ramgopal; Garnier, Gil

    2017-03-01

    Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept.

  19. Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics

    PubMed Central

    Tiwari, Sadhana; Vinchurkar, Madhuri; Rao, V. Ramgopal; Garnier, Gil

    2017-01-01

    Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept. PMID:28252113

  20. Urothelial function reconsidered: A role in urinary protein secretion

    PubMed Central

    Deng, Fang-Ming; Ding, Mingxiao; Lavker, Robert M.; Sun, Tung-Tien

    2001-01-01

    Mammalian bladder epithelium functions as an effective permeability barrier. We demonstrate here that this epithelium can also function as a secretory tissue directly involved in modifying urinary protein composition. Our data indicate that normal bovine urothelium synthesizes, as its major differentiation products, two well-known proteases: tissue-type plasminogen activator and urokinase, as well as a serine protease inhibitor, PP5. Moreover, we demonstrate that the urothelium secretes these proteins in a polarized fashion into the urine via a cAMP- and calcium-regulated pathway. Urinary plasminogen activators of ruminants are therefore urothelium derived rather then kidney derived as in some other species; this heterogeneity may have evolved in response to different physiological or dietary factors. In conjunction with our recent finding that transgenic mouse urothelium can secrete ectopically expressed human growth hormone into the urine, our data establish that normal mammalian urothelium can function not only as a permeability barrier but also as a secretor of urinary proteins that can play physiological or pathological roles in the urinary tract. PMID:11136252

  1. Moving in the Right Direction: Protein Vibrational Steering Function.

    PubMed

    Niessen, Katherine A; Xu, Mengyang; Paciaroni, Alessandro; Orecchini, Andrea; Snell, Edward H; Markelz, Andrea G

    2017-03-14

    Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calculations have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy, we find that there is a dramatic change to the vibrational directionality with inhibitor binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramolecular vibrations, and immediately resolve the inconsistency between calculations and previous measurements, which were only sensitive to the energy distribution. The biological importance of the vibrational directions versus the energy distribution is revealed by our calculations comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.

  2. Functional characterization of Clostridium difficile spore coat proteins.

    PubMed

    Permpoonpattana, Patima; Phetcharaburanin, Jutarop; Mikelsone, Anna; Dembek, Marcin; Tan, Sisareuth; Brisson, Marie-Clémence; La Ragione, Roberto; Brisson, Alain R; Fairweather, Neil; Hong, Huynh A; Cutting, Simon M

    2013-04-01

    Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H2O2. Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.

  3. Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

    PubMed Central

    Lloyd, J A; Lee, R F; Lingrel, J B

    1989-01-01

    Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator. Images PMID:2472607

  4. Functional Analysis of GLRX5 Mutants Reveals Distinct Functionalities of GLRX5 Protein.

    PubMed

    Liu, Gang; Wang, Yongwei; Anderson, Gregory J; Camaschella, Clara; Chang, Yanzhong; Nie, Guangjun

    2016-01-01

    Glutaredoxin 5 (GLRX5) is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different amino acids of the protein will lead to distinct effects on downstream Fe/S biosynthesis.

  5. Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies

    PubMed Central

    Midzak, Andrew; Papadopoulos, Vassilios

    2016-01-01

    The adrenal cortex is critical for physiological function as the central site of glucocorticoid and mineralocorticoid synthesis. It possesses a great degree of specialized compartmentalization at multiple hierarchical levels, ranging from the tissue down to the molecular levels. In this paper, we discuss this functionalization, beginning with the tissue zonation of the adrenal cortex and how this impacts steroidogenic output. We then discuss the cellular biology of steroidogenesis, placing special emphasis on the mitochondria. Mitochondria are classically known as the “powerhouses of the cell” for their central role in respiratory adenosine triphosphate synthesis, and attention is given to mitochondrial electron transport, in both the context of mitochondrial respiration and mitochondrial steroid metabolism. Building on work demonstrating functional assembly of large protein complexes in respiration, we further review research demonstrating a role for multimeric protein complexes in mitochondrial cholesterol transport, steroidogenesis, and mitochondria–endoplasmic reticulum contact. We aim to highlight with this review the shift in steroidogenic cell biology from a focus on the actions of individual proteins in isolation to the actions of protein assemblies working together to execute cellular functions. PMID:27524977

  6. Pulse Dipolar ESR and Protein Superstructures and Function

    NASA Astrophysics Data System (ADS)

    Freed, Jack

    2014-03-01

    Pulse dipolar electron-spin resonance (PDS-ESR) has emerged as a powerful methodology for the study of protein structure and function. This technology, in the form of double quantum coherence (DQC) - ESR and double-electron-electron resonance (DEER) in conjunction with site-directed spin-labeling will be described. It enables the measurement of distances and their distributions in the range of 1-9 nm between pairs of spins labeled at two sites in the protein. Many biological objects can be studied: soluble and membrane proteins, protein complexes, etc. Many sample morphologies are possible: uniform, heterogeneous, etc. thereby permitting a variety of sample types: solutions, liposomes, micelles, bicelles. Concentrations from micromolar to tens of millimolar are amenable, requiring only small amounts of biomolecules. The distances are quite accurate, so a relatively small number of them are sufficient to reveal structures and functional details. Several examples will be shown. The first is defining the protein complexes that mediate bacterial chemotaxis, which is the process whereby cells modulate their flagella-driven motility in response to environmental cues. It relies on a complex sensory apparatus composed of transmembrane receptors, histidine kinases, and coupling proteins. PDS-based models have captured key architectural features of the receptor kinase arrays and the flagellar motor, and their changes in conformation and dynamics that accompany kinase activation and motor switching. Another example will be determining the conformational states and cycling of a membrane transporter, GltPh, which is a homotrimer, in its apo, substrate-bound, and inhibitor-bound, states in membrane vesicles providing insight into its energetics. In a third example the structureless (in solution) proteins alpha-synuclein and tau, which are important in Parkinson's disease and in neurodegeneration will be described and the structures they take on in contact with membranes will be

  7. Polyethyleneimine-modified graphene oxide nanocomposites for effective protein functionalization

    NASA Astrophysics Data System (ADS)

    Weng, Yejing; Jiang, Bo; Yang, Kaiguang; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2015-08-01

    A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions.A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions. Electronic supplementary information (ESI) available: Cell viability assay, enrichment of standard glycoprotein, pretreatment and analysis of real

  8. Assessing scoring functions for protein-ligand interactions.

    PubMed

    Ferrara, Philippe; Gohlke, Holger; Price, Daniel J; Klebe, Gerhard; Brooks, Charles L

    2004-06-03

    An assessment of nine scoring functions commonly applied in docking using a set of 189 protein-ligand complexes is presented. The scoring functions include the CHARMm potential, the scoring function DrugScore, the scoring function used in AutoDock, the three scoring functions implemented in DOCK, as well as three scoring functions implemented in the CScore module in SYBYL (PMF, Gold, ChemScore). We evaluated the abilities of these scoring functions to recognize near-native configurations among a set of decoys and to rank binding affinities. Binding site decoys were generated by molecular dynamics with restraints. To investigate whether the scoring functions can also be applied for binding site detection, decoys on the protein surface were generated. The influence of the assignment of protonation states was probed by either assigning "standard" protonation states to binding site residues or adjusting protonation states according to experimental evidence. The role of solvation models in conjunction with CHARMm was explored in detail. These include a distance-dependent dielectric function, a generalized Born model, and the Poisson equation. We evaluated the effect of using a rigid receptor on the outcome of docking by generating all-pairs decoys ("cross-decoys") for six trypsin and seven HIV-1 protease complexes. The scoring functions perform well to discriminate near-native from misdocked conformations, with CHARMm, DOCK-energy, DrugScore, ChemScore, and AutoDock yielding recognition rates of around 80%. Significant degradation in performance is observed in going from decoy to cross-decoy recognition for CHARMm in the case of HIV-1 protease, whereas DrugScore and ChemScore, as well as CHARMm in the case of trypsin, show only small deterioration. In contrast, the prediction of binding affinities remains problematic for all of the scoring functions. ChemScore gives the highest correlation value with R(2) = 0.51 for the set of 189 complexes and R(2) = 0.43 for the set

  9. Upstream Design and 1D-CAE

    NASA Astrophysics Data System (ADS)

    Sawada, Hiroyuki

    Recently, engineering design environment of Japan is changing variously. Manufacturing companies are being challenged to design and bring out products that meet the diverse demands of customers and are competitive against those produced by rising countries(1). In order to keep and strengthen the competitiveness of Japanese companies, it is necessary to create new added values as well as conventional ones. It is well known that design at the early stages has a great influence on the final design solution. Therefore, design support tools for the upstream design is necessary for creating new added values. We have established a research society for 1D-CAE (1 Dimensional Computer Aided Engineering)(2), which is a general term for idea, methodology and tools applicable for the upstream design support, and discuss the concept and definition of 1D-CAE. This paper reports our discussion about 1D-CAE.

  10. Membrane proteins in four acts: function precedes structure determination.

    PubMed

    Cramer, W A; Zakharov, S D; Saif Hasan, S; Zhang, H; Baniulis, D; Zhalnina, M V; Soriano, G M; Sharma, O; Rochet, J C; Ryan, C; Whitelegge, J; Kurisu, G; Yamashita, E

    2011-12-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to

  11. Sequence of the WT1 upstream region including the Wit-1 gene

    SciTech Connect

    Gessler, M. ); Bruns, G.A.P. )

    1993-08-01

    The Wilms tumor gene WT1 encodes a Cys[sub 2]His[sub 2]-type zinc finger protein that can bind DNA and function as a transcriptional regulator. The pathological spectrum of tumorigenesis and various developmental defects produced by different WT1 alteration suggests that WT1 controls a number of subsequent effector genes. To define the role of WT1 in these developmental processes it will be important to elucidate mechanisms that govern expression of WT1 itself. To facilitate mapping of the WT1 promoter region and 5[prime] control elements the authors have determined the sequence upstream of the WT1 transcription unit. This includes the Wit-1 gene that is transcribed in the opposite direction. 11 refs., 3 figs.

  12. Admissible upstream conditions for slender compressible vortices

    NASA Technical Reports Server (NTRS)

    Liu, C. H.; Krause, E.; Menne, S.

    1986-01-01

    The influence of the compressibility on the flow in slender vortices is being studied. The dependence of the breakdown of the slender-vortex approximation on the upstream conditions is demonstrated for various Reynolds numbers and Mach numbers. Compatibility conditions, which have to be satisfied if the vortex is to remain slender, are discussed in detail. The general discussions are supplemented by several sample calculations.

  13. Exopolysaccharides modify functional properties of whey protein concentrate.

    PubMed

    Deep, G; Hassan, A N; Metzger, L

    2012-11-01

    The objective of this research was to produce whey protein concentrate (WPC) with modified functionality using exopolysaccharide- (EPS) producing cultures. Two different EPS-producing cultures, Lactococcus lactis ssp. cremoris JFR and Streptococcus thermophilus, producing EPS1 and EPS2 respectively, were used in this study. One EPS-nonproducing commercial cheese culture (DVS 850; Chr. Hansen, Milwaukee, WI) was used as the control. Reconstituted sweet whey powder was used in this study to eliminate variations from fresh whey. Cultures grown overnight in reconstituted WPC (10% wt/vol) were added, directly or after overnight cooling (cooled EPS), at 2% (wt/vol) to 6% (wt/wt) solution of reconstituted whey. Whey was then high-temperature, short-time pasteurized at 75 °C for 35s and ultrafiltered to a volume reduction factor of 5. Ultrafiltered whey (retentate) was spray dried at inlet and outlet air temperatures of 200 and 90 °C, respectively, to obtain WPC. In general, the solubility of WPC was higher at pH 7 than at pH 3. Whey protein concentrate containing EPS2 exhibited higher protein solubility than did WPC containing no EPS. Also, the presence of EPS in WPC decreased protein denaturation. The emulsifying ability of WPC containing EPS was higher than that in control. Addition of EPS to WPC significantly enhanced its gelling ability. Foam overrun and hydrophobicity of WPC were not affected by addition of EPS. In conclusion, data obtained from this study show that EPS modify WPC functionality. The extent of modification depends on the type of EPS. Cooling of culture containing EPS before its addition to whey further reduced WPC protein denaturation and increased its solubility at pH 7 and gel hardness.

  14. Heat-induced whey protein gels: protein-protein interactions and functional properties.

    PubMed

    Havea, Palatasa; Watkinson, Philip; Kuhn-Sherlock, Barbara

    2009-02-25

    Heat-induced gelation (80 degrees C for 30 min or 85 degrees C for 60 min) of whey protein concentrate (WPC) solutions was studied using small deformation dynamic rheology, small and large deformation compression, and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (15% w/w, pH 6.9) were prepared by dispersing WPC powder in water (control), 1% (w/w) sodium dodecyl sulfate (SDS) solution, and N-ethylmaleimide (NEM) solution at a protein/NEM molar ratio of 1:1 or in 10 mM dithiothreitol (DTT) solution. PAGE analyses showed that the heat treatment of control solutions contained both disulfide and non-covalent linkages between denatured protein molecules. Only disulfide linkages were formed in heated SDS-WPC solutions, whereas only non-covalent linkages were formed in DTT-WPC and NEM-WPC solutions during heating. In heated NEM-WPC solutions, the pre-existing disulfide linkages remained unaltered. Small deformation rheology measurements showed that the storage modulus (G') values, compared with those of the control WPC gels (approximately 14000 Pa), were 3 times less for the SDS-WPC gels (approximately 4000 Pa), double for the NEM-WPC gels (approximately 24000 Pa), and even higher for the DTT-WPC gels (approximately 30000 Pa). Compression tests suggested that the rubberiness (fracture strain) of the WPC gels increased as the degree of disulfide linkages within the gels increased, whereas the stiffness (modulus) of the gels increased as the degree of non-covalent associations among the denatured protein molecules increased.

  15. A novel neural response algorithm for protein function prediction

    PubMed Central

    2012-01-01

    Background Large amounts of data are being generated by high-throughput genome sequencing methods. But the rate of the experimental functional characterization falls far behind. To fill the gap between the number of sequences and their annotations, fast and accurate automated annotation methods are required. Many methods, such as GOblet, GOFigure, and Gotcha, are designed based on the BLAST search. Unfortunately, the sequence coverage of these methods is low as they cannot detect the remote homologues. Adding to this, the lack of annotation specificity advocates the need to improve automated protein function prediction. Results We designed a novel automated protein functional assignment method based on the neural response algorithm, which simulates the neuronal behavior of the visual cortex in the human brain. Firstly, we predict the most similar target protein for a given query protein and thereby assign its GO term to the query sequence. When assessed on test set, our method ranked the actual leaf GO term among the top 5 probable GO terms with accuracy of 86.93%. Conclusions The proposed algorithm is the first instance of neural response algorithm being used in the biological domain. The use of HMM profiles along with the secondary structure information to define the neural response gives our method an edge over other available methods on annotation accuracy. Results of the 5-fold cross validation and the comparison with PFP and FFPred servers indicate the prominent performance by our method. The program, the dataset, and help files are available at http://www.jjwanglab.org/NRProF/. PMID:23046521

  16. Functional aspects of membrane association of reggie/flotillin proteins.

    PubMed

    Banning, Antje; Tomasovic, Ana; Tikkanen, Ritva

    2011-12-01

    Flotillin-2 and flotillin-1, also called reggie-1 and reggie-2, are ubiquitously expressed and highly conserved proteins. Originally, they were described as neuronal regeneration proteins, but they appear to function in a wide variety of cellular processes, such as membrane receptor signaling, endocytosis, phagocytosis and cell adhesion. The molecular details of the function of flotillins in these processes have only been partially clarified. Flotillins are associated with cholesterol and sphingolipid enriched membrane microdomains known as rafts, and some findings even suggest that they define their own kind of a microdomain. The mechanism of the membrane association of flotillins appears to rely mainly on acylation (myristoylation and/or palmitoylation), localizing flotillins onto the cytosolic side of the membranes, whereas no transmembrane domains are present. In addition, flotillins show a strong tendency to form homo- and hetero-oligomers with each other. In this review, we will summarize the recent findings on the function of flotillins and discuss the mechanisms that might regulate their function, such as membrane association, oligomerization and phosphorylation.

  17. The importance of slow motions for protein functional loops.

    PubMed

    Skliros, Aris; Zimmermann, Michael T; Chakraborty, Debkanta; Saraswathi, Saras; Katebi, Ataur R; Leelananda, Sumudu P; Kloczkowski, Andrzej; Jernigan, Robert L

    2012-02-07

    Loops in proteins that connect secondary structures such as alpha-helix and beta-sheet, are often on the surface and may play a critical role in some functions of a protein. The mobility of loops is central for the motional freedom and flexibility requirements of active-site loops and may play a critical role for some functions. The structures and behaviors of loops have not been studied much in the context of the whole structure and its overall motions, especially how these might be coupled. Here we investigate loop motions by using coarse-grained structures (C(α) atoms only) to solve the motions of the system by applying Lagrange equations with elastic network models to learn about which loops move in an independent fashion and which move in coordination with domain motions, faster and slower, respectively. The normal modes of the system are calculated using eigen-decomposition of the stiffness matrix. The contribution of individual modes and groups of modes is investigated for their effects on all residues in each loop by using Fourier analyses. Our results indicate overall that the motions of functional sets of loops behave in similar ways as the whole structure. But overall only a relatively few loops move in coordination with the dominant slow modes of motion, and these are often closely related to function.

  18. Decreased function of survival motor neuron protein impairs endocytic pathways

    PubMed Central

    Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S.; O’Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C. Q.; Cook, Steven J.; Poulogiannis, George; Atwood, Walter J.; Hall, David H.; Hart, Anne C.

    2016-01-01

    Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

  19. STRING: a database of predicted functional associations between proteins.

    PubMed

    von Mering, Christian; Huynen, Martijn; Jaeggi, Daniel; Schmidt, Steffen; Bork, Peer; Snel, Berend

    2003-01-01

    Functional links between proteins can often be inferred from genomic associations between the genes that encode them: groups of genes that are required for the same function tend to show similar species coverage, are often located in close proximity on the genome (in prokaryotes), and tend to be involved in gene-fusion events. The database STRING is a precomputed global resource for the exploration and analysis of these associations. Since the three types of evidence differ conceptually, and the number of predicted interactions is very large, it is essential to be able to assess and compare the significance of individual predictions. Thus, STRING contains a unique scoring-framework based on benchmarks of the different types of associations against a common reference set, integrated in a single confidence score per prediction. The graphical representation of the network of inferred, weighted protein interactions provides a high-level view of functional linkage, facilitating the analysis of modularity in biological processes. STRING is updated continuously, and currently contains 261 033 orthologs in 89 fully sequenced genomes. The database predicts functional interactions at an expected level of accuracy of at least 80% for more than half of the genes; it is online at http://www.bork.embl-heidelberg.de/STRING/.

  20. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    -synthetic acetylation for the chaperone function of HMGB1 protein. The presence of an acetyl groups at Lys 2 decreases strongly the stimulating effect of the protein in the stepwise salt dialysis experiment and the same tendency persisted in the dialysis free experiment.

  1. Functional interactions between a glutamine synthetase promoter and MYB proteins.

    PubMed

    Gómez-Maldonado, Josefa; Avila, Concepción; Torre, Fernando; Cañas, Rafael; Cánovas, Francisco M; Campbell, Malcolm M

    2004-08-01

    In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

  2. Human PIEZO1 Ion Channel Functions as a Split Protein

    PubMed Central

    Bae, Chilman; Suchyna, Thomas M.; Ziegler, Lynn; Sachs, Frederick; Gottlieb, Philip A.

    2016-01-01

    PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. PMID:26963637

  3. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  4. EXTRACELLULAR HEAT SHOCK PROTEINS: A NEW LOCATION, A NEW FUNCTION

    PubMed Central

    De Maio, Antonio; Vazquez, Daniel

    2015-01-01

    The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp. PMID:23807250

  5. Fragile X syndrome: From protein function to therapy.

    PubMed

    Bagni, Claudia; Oostra, Ben A

    2013-11-01

    Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism. The FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The disease is a result of lack of expression of the fragile X mental retardation protein leading to severe symptoms, including intellectual disability, hyperactivity, and autistic-like behavior. The FMR1 protein (FMRP) has a number of functions. The translational dysregulation of a subset of mRNAs targeted by FMRP is probably the major contribution to FXS. FMRP is also involved in mRNA transport to synapses where protein synthesis occurs. For some FMRP-bound mRNAs, FMRP is a direct modulator of mRNA stability either by sustaining or preventing mRNA decay. Increased knowledge about the role of FMRP has led to the identification of potential treatments for fragile X syndrome that were often tested first in the different animal models. This review gives an overview about the present knowledge of the function of FMRP and the therapeutic strategies in mouse and man.

  6. Fission Yeast CSL Proteins Function as Transcription Factors

    PubMed Central

    Oravcová, Martina; Teska, Mikoláš; Půta, František; Folk, Petr; Převorovský, Martin

    2013-01-01

    Background Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members. Methodology/Principal Findings Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. Conclusions/Significance Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family. PMID:23555033

  7. SNAP-25, a Known Presynaptic Protein with Emerging Postsynaptic Functions

    PubMed Central

    Antonucci, Flavia; Corradini, Irene; Fossati, Giuliana; Tomasoni, Romana; Menna, Elisabetta; Matteoli, Michela

    2016-01-01

    A hallmark of synaptic specializations is their dependence on highly organized complexes of proteins that interact with each other. The loss or modification of key synaptic proteins directly affects the properties of such networks, ultimately impacting synaptic function. SNAP-25 is a component of the SNARE complex, which is central to synaptic vesicle exocytosis, and, by directly interacting with different calcium channels subunits, it negatively modulates neuronal voltage-gated calcium channels, thus regulating intracellular calcium dynamics. The SNAP-25 gene has been associated with distinct brain diseases, including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and bipolar disorder, indicating that the protein may act as a shared biological substrate among different “synaptopathies”. The mechanisms by which alterations in SNAP-25 may concur to these psychiatric diseases are still undefined, although alterations in neurotransmitter release have been indicated as potential causative processes. This review summarizes recent work showing that SNAP-25 not only controls exo/endocytic processes at the presynaptic terminal, but also regulates postsynaptic receptor trafficking, spine morphogenesis, and plasticity, thus opening the possibility that SNAP-25 defects may contribute to psychiatric diseases by impacting not only presynaptic but also postsynaptic functions. PMID:27047369

  8. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  9. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing*

    PubMed Central

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-01-01

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a 13C-labeled retinal cofactor and extensively 13C-15N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications. PMID:26405032

  10. Polymodal sensory function of the Caenorhabditis elegans OCR-2 channel arises from distinct intrinsic determinants within the protein and is selectively conserved in mammalian TRPV proteins.

    PubMed

    Sokolchik, Irina; Tanabe, Takahiro; Baldi, Pierre F; Sze, Ji Ying

    2005-01-26

    Caenorhabditis elegans OCR-2 (OSM-9 and capsaicin receptor-related) is a TRPV (vanilloid subfamily of transient receptor potential channel) protein that regulates serotonin (5-HT) biosynthesis in chemosensory neurons and also mediates olfactory and osmotic sensation. Here, we identify the molecular basis for the polymodal function of OCR-2 in its native cellular environment. We show that OCR-2 function in 5-HT production and osmotic sensing is governed by its N-terminal region upstream of the ankyrin repeats domain, but the diacetyl sensitivity is mediated by independent mechanisms. The ocr-2(yz5) mutation results in a glycine-to-glutamate substitution (G36E) within the N-terminal region. The G36E substitution causes dramatic downregulation of 5-HT synthesis in the ADF neurons, eliminates osmosensation mediated by the ASH neurons, but does not affect the response to the odorant diacetyl mediated by the AWA neurons. Conversely, wild-type sequence of the N-terminal segment confers osmotic sensitivity and upregulation of 5-HT production to a normally insensitive C. elegans homolog, OCR-4, but this chimeric channel does not respond to diacetyl stimuli. Furthermore, expression of either the mouse or human TRPV2 gene under the ocr-2 promoter can substantially restore 5-HT biosynthesis in ocr-2-null mutants but cannot improve the deficits in osmotic or olfactory sensation, suggesting that TRPV2 can substitute for the role of OCR-2 only in serotonergic neurons. Thus, different sensory functions of OCR-2 arise from separable intrinsic determinants, and specific functional properties of TRPV channel proteins may be selectively conserved across phyla.

  11. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins.

  12. Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

    PubMed

    Ahir, Bhavesh K; Pratten, Margaret K

    2014-01-01

    Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

  13. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    PubMed Central

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  14. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

    PubMed

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-08-02

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

  15. Pathway logic modeling of protein functional domains in signal transduction.

    PubMed

    Talcott, C; Eker, S; Knapp, M; Lincoln, P; Laderoute, K

    2004-01-01

    Protein functional domains (PFDs) are consensus sequences within signaling molecules that recognize and assemble other signaling components into complexes. Here we describe the application of an approach called Pathway Logic to the symbolic modeling signal transduction networks at the level of PFDs. These models are developed using Maude, a symbolic language founded on rewriting logic. Models can be queried (analyzed) using the execution, search and model-checking tools of Maude. We show how signal transduction processes can be modeled using Maude at very different levels of abstraction involving either an overall state of a protein or its PFDs and their interactions. The key insight for the latter is our algebraic representation of binding interactions as a graph.

  16. Combining heterogeneous data sources for accurate functional annotation of proteins

    PubMed Central

    2013-01-01

    Combining heterogeneous sources of data is essential for accurate prediction of protein function. The task is complicated by the fact that while sequence-based features can be readily compared across species, most other data are species-specific. In this paper, we present a multi-view extension to GOstruct, a structured-output framework for function annotation of proteins. The extended framework can learn from disparate data sources, with each data source provided to the framework in the form of a kernel. Our empirical results demonstrate that the multi-view framework is able to utilize all available information, yielding better performance than sequence-based models trained across species and models trained from collections of data within a given species. This version of GOstruct participated in the recent Critical Assessment of Functional Annotations (CAFA) challenge; since then we have significantly improved the natural language processing component of the method, which now provides performance that is on par with that provided by sequence information. The GOstruct framework is available for download at http://strut.sourceforge.net. PMID:23514123

  17. A functional protein pore with a "retro" transmembrane domain.

    PubMed Central

    Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H.

    1999-01-01

    Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif. PMID:10386875

  18. Prion Protein Expression and Functional Importance in Skeletal Muscle

    PubMed Central

    Smith, Jeffrey D.; Moylan, Jennifer S.; Hardin, Brian J.; Chambers, Melissa A.; Estus, Steven; Telling, Glenn C.

    2011-01-01

    Abstract Skeletal muscle expresses prion protein (PrP) that buffers oxidant activity in neurons. Aims We hypothesize that PrP deficiency would increase oxidant activity in skeletal muscle and alter redox-sensitive functions, including contraction and glucose uptake. We used real-time polymerase chain reaction and Western blot analysis to measure PrP mRNA and protein in human diaphragm, five murine muscles, and muscle-derived C2C12 cells. Effects of PrP deficiency were tested by comparing PrP-deficient mice versus wild-type mice and morpholino-knockdown versus vehicle-treated myotubes. Oxidant activity (dichlorofluorescin oxidation) and specific force were measured in murine diaphragm fiber bundles. Results PrP content differs among mouse muscles (gastrocnemius>extensor digitorum longus, EDL>tibialis anterior, TA; soleus>diaphragm) as does glycosylation (di-, mono-, nonglycosylated; gastrocnemius, EDL, TA=60%, 30%, 10%; soleus, 30%, 40%, 30%; diaphragm, 30%, 30%, 40%). PrP is predominantly di-glycosylated in human diaphragm. PrP deficiency decreases body weight (15%) and EDL mass (9%); increases cytosolic oxidant activity (fiber bundles, 36%; C2C12 myotubes, 7%); and depresses specific force (12%) in adult (8–12 mos) but not adolescent (2 mos) mice. Innovation This study is the first to directly assess a role of prion protein in skeletal muscle function. Conclusions PrP content varies among murine skeletal muscles and is essential for maintaining normal redox homeostasis, muscle size, and contractile function in adult animals. Antioxid. Redox Signal. 15, 2465—2475. PMID:21453198

  19. Context-based retrieval of functional modules in protein-protein interaction networks.

    PubMed

    Dobay, Maria Pamela; Stertz, Silke; Delorenzi, Mauro

    2017-03-27

    Various techniques have been developed for identifying the most probable interactants of a protein under a given biological context. In this article, we dissect the effects of the choice of the protein-protein interaction network (PPI) and the manipulation of PPI settings on the network neighborhood of the influenza A virus (IAV) network, as well as hits in genome-wide small interfering RNA screen results for IAV host factors. We investigate the potential of context filtering, which uses text mining evidence linked to PPI edges, as a complement to the edge confidence scores typically provided in PPIs for filtering, for obtaining more biologically relevant network neighborhoods. Here, we estimate the maximum performance of context filtering to isolate a Kyoto Encyclopedia of Genes and Genomes (KEGG) network Ki from a union of KEGG networks and its network neighborhood. The work gives insights on the use of human PPIs in network neighborhood approaches for functional inference.

  20. The retinoblastoma protein: functions beyond the G1-S regulator.

    PubMed

    Uchida, Chiharu

    2012-12-01

    Retinoblastoma protein (pRB) is functionally inactivated in a large number of tumors including retinoblastoma, osteosarcoma, small-cell lung carcinoma, as well as bladder, breast and prostate cancers. The best known role of pRB in preventing cancer is inhibition of cell cycle progression by controlling the exit from the cell cycle into G0/G1. In addition, increasing evidence has suggested that pRB has important roles in DNA replication during S phase and G2/M transition. The tumor suppressor function of pRB has also been demonstrated by directly promoting differentiation via cell cycle exit with specific gene expression. Inactivation of pRB function during these cell cycle phases leads to dysregulated cell proliferation and/or chromosomal instability, which are strongly linked to cancer development. Thus pRB plays important roles through multiple functions in determining cell fate, i.e., normal growth/death and differentiation, or tumor formation. Therapeutic intervention by reactivation of pRB function would be expected to be an effective treatment against various cancers.

  1. Reversible lysine modification on proteins by using functionalized boronic acids.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

  2. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  3. Biofield-effect protein-sensor: Plasma functionalization of polyaniline, protein immobilization, and sensing mechanism

    NASA Astrophysics Data System (ADS)

    Cho, Chae-Ryong; Lee, Hyun-Uk; Ahn, Kyun; Jeong, Se-Young; Choi, Jun-Hee; Kim, Jinwoo; Cho, Jiung

    2014-06-01

    We report the fabrication of a biofield-effect protein-sensor (BioFEP) based on atmospheric-pressure plasma (AP) treatment of a conducting polyaniline (PANI) film. Successive H2 and O2 AP (OHAP) treatment generated dominant hydrophilic -OH and O=CO- functional groups on the PANI film surface, which served as strong binding sites to immobilize bovine serum albumin (BSA) protein molecules. The output current changes of the BioFEP as a function of BSA concentration were obtained. The resistance of the OHAP surface could be sensitively increased from 2.5 × 108 Ω to 2.0 × 1012 Ω with increasing BSA concentrations in the range of 0.025-4 μg/ml. The results suggest that the method is a simple and cost-effective tool to determine the concentration of BSA by measuring electrical resistance.

  4. Interaction between nanoparticles and cytokine proteins: impact on protein and particle functionality

    NASA Astrophysics Data System (ADS)

    Brown, David M.; Dickson, Claire; Duncan, Paul; Al-Attili, Furat; Stone, Vicki

    2010-05-01

    There is increased use of nanomaterials in many applications due to their unique properties, such as their high surface area and surface reactivity. However, the potential health effects to workers, consumers and the environment exposed to nanoparticles (NPs) is unknown. The aim of this study was to investigate whether NPs which may enter the body could adsorb proteins and whether this interaction affects both the particle and the protein function. The cytokines IL-8 and TNF-α were adsorbed significantly more by 14 nm carbon black (CB) compared with a similar dose of 260 nm CB. Uncoated 14 nm CB particles produced a significant increase in intracellular calcium [Ca2 + ]i which was greater than a similar mass dose of 260 nm CB. The 260 nm CB produced an increase in ICAM-1 expression in A549 epithelial cells at a comparable dose of 14 nm CB, and after coating with TNF-α 260 nm CB produced significantly more ICAM-1 expression compared with control cells. TNF-α bound to 14 nm CB induced a level of ICAM-1 expression that was no greater than the control level, suggesting that the TNF-α activity may be inhibited. These results suggest that NP-protein interaction results both in a decrease in protein function and particle activity in the cellular assays tested and this is currently being investigated.

  5. Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

    PubMed Central

    Tang, Hai-Bo; Lu, Zhuan-Ling; Wei, Xian-Kai; Zhong, Tao-Zhen; Zhong, Yi-Zhi; Ouyang, Ling-Xuan; Luo, Yang; Xing, Xing-Wei; Liao, Fang; Peng, Ke-Ke; Deng, Chao-Qian; Minamoto, Nobuyuki; Luo, Ting Rong

    2016-01-01

    Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4. PMID:27456665

  6. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  7. A First Line of Stress Defense: Small Heat Shock Proteins and their function in protein homeostasis

    PubMed Central

    Haslbeck, Martin; Vierling, Elizabeth

    2015-01-01

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. To maintain protein homeostasis, sHsps complex with a variety of nonnative proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation. In vertebrates they act to maintain the clarity of the eye lens, and in humans sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42 kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or coassembly between different sHsps in the same cellular compartment adds an underexplored level of complexity to sHsp structure and function. PMID:25681016

  8. A first line of stress defense: small heat shock proteins and their function in protein homeostasis.

    PubMed

    Haslbeck, Martin; Vierling, Elizabeth

    2015-04-10

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. sHsps complex with a variety of non-native proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation in order to maintain protein homeostasis. In vertebrates, they act to maintain the clarity of the eye lens, and in humans, sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel-like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or co-assembly between different sHsps in the same cellular compartment add an underexplored level of complexity to sHsp structure and function.

  9. UBXD