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Sample records for protein functions upstream

  1. The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

    PubMed

    Wang, Wenjing; Liu, Guanshan; Niu, Haixia; Timko, Michael P; Zhang, Hongbo

    2014-05-01

    Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

  2. The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90)

    PubMed Central

    Zhang, Hongbo

    2014-01-01

    Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes. PMID:24604735

  3. 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

  4. The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance in Neurospora crassa

    PubMed Central

    Jones, Carol A.; Greer-Phillips, Suzanne E.

    2007-01-01

    Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Δrrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Δrrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Δrrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Δrrg-1 strains. OS-2-phosphate levels are also low in Δrrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1D921N allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway. PMID:17392518

  5. 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

  6. The Arabidopsis Mitogen-Activated Protein Kinase Kinase Kinase 20 (MKKK20) Acts Upstream of MKK3 and MPK18 in Two Separate Signaling Pathways Involved in Root Microtubule Functions.

    PubMed

    Benhamman, Rachid; Bai, Fangwen; Drory, Samuel B; Loubert-Hudon, Audrey; Ellis, Brian; Matton, Daniel P

    2017-01-01

    Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein-protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.

  7. Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

    PubMed Central

    Vignais, M L; Woudt, L P; Wassenaar, G M; Mager, W H; Sentenac, A; Planta, R J

    1987-01-01

    Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:3301327

  8. 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.

  9. 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.

  10. Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus

    PubMed Central

    Fromme, Tobias; Reichwald, Kathrin; Platzer, Matthias; Li, Xing-Sheng; Klingenspor, Martin

    2007-01-01

    Background Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents. Results By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays. Conclusion Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence

  11. NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways.

    PubMed

    Li, Jie; Brown, Gemma; Ailion, Michael; Lee, Samuel; Thomas, James H

    2004-11-01

    Mutations in the human NPC1 gene cause most cases of Niemann-Pick type C (NP-C) disease, a fatal autosomal recessive neurodegenerative disorder. NPC1 is implicated in intracellular trafficking of cholesterol and glycolipids, but its exact function remains unclear. The C. elegans genome contains two homologs of NPC1, ncr-1 and ncr-2, and an ncr-2; ncr-1 double deletion mutant forms dauer larvae constitutively (Daf-c). We have analyzed the phenotypes of ncr single and double mutants in detail, and determined the ncr gene expression patterns. We find that the ncr genes function in a hormonal branch of the dauer formation pathway upstream of daf-9 and daf-12, which encode a cytochrome P450 enzyme and a nuclear hormone receptor, respectively. ncr-1 is expressed broadly in tissues with high levels of cholesterol, whereas expression of ncr-2 is restricted to a few cells. Both Ncr genes are expressed in the XXX cells, which are implicated in regulating dauer formation via the daf-9 pathway. Only the ncr-1 mutant is hypersensitive to cholesterol deprivation and to progesterone, an inhibitor of intracellular cholesterol trafficking. Our results support the hypothesis that ncr-1 and ncr-2 are involved in intracellular cholesterol processing in C. elegans, and that a sterol-signaling defect is responsible for the Daf-c phenotype of the ncr-2; ncr-1 mutant.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

    PubMed Central

    Vijayaraj, Preethi; Kröger, Cornelia; Reuter, Ursula; Windoffer, Reinhard; Leube, Rudolf E.

    2009-01-01

    Keratin intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which affects cytoarchitecture, cell growth, survival, and organelle transport. However, owing to redundancy, the global function of keratins has not been defined in full. Using a targeted gene deletion strategy, we generated transgenic mice lacking the entire keratin multiprotein family. In this study, we report that without keratins, embryonic epithelia suffer no cytolysis and maintain apical polarity but display mislocalized desmosomes. All keratin-null embryos die from severe growth retardation at embryonic day 9.5. We find that GLUT1 and -3 are mislocalized from the apical plasma membrane in embryonic epithelia, which subsequently activates the energy sensor adenosine monophosphate kinase (AMPK). Analysis of the mammalian target of rapamycin (mTOR) pathway reveals that AMPK induction activates Raptor, repressing protein biosynthesis through mTORC1's downstream targets S6 kinase and 4E-binding protein 1. Our findings demonstrate a novel keratin function upstream of mTOR signaling via GLUT localization and have implications for pathomechanisms and therapy approaches for keratin disorders and the analysis of other gene families. PMID:19841136

  18. 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

  19. Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1).

    PubMed Central

    Sully, Gareth; Dean, Jonathan L E; Wait, Robin; Rawlinson, Lesley; Santalucia, Tomas; Saklatvala, Jeremy; Clark, Andrew R

    2004-01-01

    COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present. PMID:14594446

  20. Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression

    PubMed Central

    Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L.; Feng, Ying

    2013-01-01

    Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3′splice site, and the other involves an exonic splicing silencer (ESS) situated 3′ to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator. PMID:23818605

  1. Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression.

    PubMed

    Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L; Feng, Ying

    2013-07-16

    Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem-loop structure located downstream of the 3'splice site, and the other involves an exonic splicing silencer (ESS) situated 3' to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.

  2. Far Upstream Element-Binding Protein 1 Binds the 3' Untranslated Region of PKD2 and Suppresses Its Translation.

    PubMed

    Zheng, Wang; Shen, Fan; Hu, Ruikun; Roy, Birbickram; Yang, JungWoo; Wang, Qian; Zhang, Fan; King, Jennifer C; Sergi, Consolato; Liu, Song-Mei; Cordat, Emmanuelle; Tang, Jingfeng; Cao, Ying; Ali, Declan; Chen, Xing-Zhen

    2016-09-01

    Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD. Copyright © 2016 by the American Society of Nephrology.

  3. 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

  4. 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.

  5. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits

    PubMed Central

    Ferguson, Shawn M.; Raimondi, Andrea; Paradise, Summer; Shen, Hongying; Mesaki, Kumi; Ferguson, Agnes; Destaing, Olivier; Ko, Genevieve; Takasaki, Junko; Cremona, Ottavio; O’ Toole, Eileen; De Camilli, Pietro

    2010-01-01

    SUMMARY The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin nucleating proteins, actin and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wildtype cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin, and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin’s endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity. PMID:20059951

  6. 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

  7. 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

  8. 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

  9. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    SciTech Connect

    Craft, Willie Warren; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2005-10-10

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F{sub 0}, and proteolytically cleaved into the mature F{sub 1} and F{sub 2} heterodimer, following an HDLVDGVK{sub 109} motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK{sub 109} motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK{sub 109} motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein.

  10. 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

  11. 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

  12. 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…

  13. 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…

  14. The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein.

    PubMed

    Kwon, H; Green, M R

    1994-12-02

    The accurate transcription of human rRNA genes by RNA polymerase I requires two transcription factors, upstream binding factor (UBF) and promoter selectivity factor (SL1). Human SL1 (hSL1) is a multisubunit complex, one of whose components is TATA box-binding protein (TBP). hSL1 binds to the core region of the rRNA promoter, but does so inefficiently in the absence of human UBF (hUBF). hUBF interacts with the upstream control element of the rRNA promoter and facilitates binding of hSL1. The molecular basis by which hUBF increases binding of hSL1 remains to be elucidated. In this report, we use an immobilized protein binding assay to identify and purify a 95-kDa TBP-binding polypeptide. Microsequence analysis reveals that the 95-kDa TBP-binding protein is hUBF. We show that hUBF is stably associated with TBP and is present in large TBP-containing complexes. Our results indicate that the cooperative binding of hUBF and hSL1 on the rRNA promoter is mediated by direct interaction between hUBF and TBP. We also provide evidence that hUBF associates with TFIID, a TBP-containing RNA polymerase II transcription factor.

  15. The Martian escape rate as a function of upstream solar conditions

    NASA Astrophysics Data System (ADS)

    Ramstad, R.; Barabash, S.; Futaana, Y.; Nilsson, H.; Holmstrom, M.

    2014-12-01

    We investigate potential factors for influence on the Martian heavy ion escape rate (Q) by integrating Mars Express ASPERA-3/IMA heavy ion flux measurements in the Martian tail, taken at similar (binned) solar wind density (n), velocity (v) and EUV radiation flux (FEUV) upstream conditions. In the best sampled cases, with v and FEUV constrained, we find a statistically significant decrease in heavy ion escape rate with increased solar wind density. An empirical-analytical model for atmospheric escape is developed by fitting calculated escape rates to all sufficiently sampled solar conditions, indicating an overall negative dependence on solar wind density.

  16. [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.

  17. 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.

  18. Comparative genomics reveals a functional thyroid-specific element in the far upstream region of the PAX8 gene

    PubMed Central

    2010-01-01

    Background The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. Results We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. Conclusions Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Differences in brain gene transcription profiles advocate for an important role of cognitive function in upstream migration and water obstacles crossing in European eel.

    PubMed

    Podgorniak, Tomasz; Milan, Massimo; Pujolar, Jose Marti; Maes, Gregory E; Bargelloni, Luca; De Oliveira, Eric; Pierron, Fabien; Daverat, Francoise

    2015-05-12

    European eel is a panmictic species, whose decline has been recorded since the last 20 years. Among human-induced environmental factors of decline, the impact of water dams during species migration is questioned. The main issue of this study was to pinpoint phenotypic traits that predisposed glass eels to successful passage by water barriers. The approach of the study was individual-centred and without any a priori hypothesis on traits involved in the putative obstacles selective pressure. We analyzed the transcription level of 14,913 genes. Transcriptome analysis of three tissues (brain, liver and muscle) from individuals sampled on three successive forebays separated by water obstacles indicated different gene transcription profiles in brain between the two upstream forebays. No differences in gene transcription levels were observed in liver and muscle samples among segments. A total of 26 genes were differentially transcribed in brain. These genes encode for, among others, keratins, cytokeratins, calcium binding proteins (S100 family), cofilin, calmodulin, claudin and thy-1 membrane glycoprotein. The functional analysis of these genes highlighted a putative role of cytoskeletal dynamics and synaptic plasticity in fish upstream migration. Synaptic connections in brain are solicited while eels are climbing the obstacles with poorly designed fishways. Successful passage by such barriers can be related to spatial learning and spatial orientation abilities when fish is out of the water.

  4. CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of hypC and Acts as a Repressor of Its Transcription in the Cyanobacterium Nostoc sp. Strain PCC 7120▿ †

    PubMed Central

    Agervald, Åsa; Zhang, Xiaohui; Stensjö, Karin; Devine, Ellenor; Lindblad, Peter

    2010-01-01

    The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that calA is cotranscribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on hypC. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect. In silico studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed. PMID:20023111

  5. 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

  6. From C-Reactive Protein to Interleukin-6 to Interleukin-1: Moving Upstream To Identify Novel Targets for Atheroprotection.

    PubMed

    Ridker, Paul M

    2016-01-08

    Plasma levels of the inflammatory biomarker high-sensitivity C-reactive protein (hsCRP) predict vascular risk with an effect estimate as large as that of total or high-density lipoprotein cholesterol. Further, randomized trial data addressing hsCRP have been central to understanding the anti-inflammatory effects of statin therapy and have consistently demonstrated on-treatment hsCRP levels to be as powerful a predictor of residual cardiovascular risk as on-treatment levels of low-density lipoprotein cholesterol. Yet, although hsCRP is clinically useful as a biomarker for risk prediction, most mechanistic studies suggest that CRP itself is unlikely to be a target for intervention. Moving upstream in the inflammatory cascade from CRP to interleukin (IL)-6 to IL-1 provides novel therapeutic opportunities for atheroprotection that focus on the central IL-6 signaling system and ultimately on inhibition of the IL-1β-producing NOD-like receptor family pyrin domain containing 3 inflammasome. Cholesterol crystals, neutrophil extracellular traps, atheroprone flow, and local tissue hypoxia activate the NOD-like receptor family pyrin domain containing 3 inflammasome. As such, a unifying concept of hsCRP as a downstream surrogate biomarker for upstream IL-1β activity has emerged. From a therapeutic perspective, small ischemia studies show reductions in acute-phase hsCRP production with the IL-1 receptor antagonist anakinra and the IL-6 receptor blocker tocilizumab. A phase IIb study conducted among diabetic patients at high vascular risk indicates that canakinumab, a human monoclonal antibody that targets IL-1β, markedly reduces plasma levels of IL-6, hsCRP, and fibrinogen with little change in atherogenic lipids. Canakinumab in now being tested as a method to prevent recurrent cardiovascular events in a randomized trial of 10 065 post-myocardial infarction patients with elevated hsCRP that is fully enrolled and due to complete in 2017. Clinical trials using alternative anti

  7. ZmCPK11 is involved in abscisic acid-induced antioxidant defence and functions upstream of ZmMPK5 in abscisic acid signalling in maize

    PubMed Central

    Jiang, Mingyi

    2013-01-01

    Calcium-dependent protein kinases (CDPKs) have been shown to be involved in abscisic acid (ABA)-mediated physiological processes, including seed germination, post-germination growth, stomatal movement, and plant stress tolerance. However, it is not clear whether CDPKs are involved in ABA-induced antioxidant defence. In the present study, the role of the maize CDPK ZmCPK11 in ABA-induced antioxidant defence and the relationship between ZmCPK11 and ZmMPK5, a maize ABA-activated mitogen-activated protein kinase (MAPK), in ABA signalling were investigated. Treatments with ABA and H2O2 induced the expression of ZmCPK11 and increased the activity of ZmCPK11, while H2O2 was required for the ABA-induced increases in the expression and the activity of ZmCPK11. The transient gene expression analysis and the transient RNA interference (RNAi) test in protoplasts showed that ZmCPK11 is involved in ABA-induced up-regulation of the expression and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), and in the production of H2O2. Further, ZmCPK11 was shown to be required for the up-regulation of the expression and the activity of ZmMPK5 in ABA signalling, but ZmMPK5 had very little effect on the ABA-induced up-regulation of the expression and the activity of ZmCPK11. Moreover, the transient gene expression analysis in combination with the transient RNAi test in protoplasts showed that ZmCPK11 acts upstream of ZmMPK5 to regulate the activities of antioxidant enzymes. These results indicate that ZmCPK11 is involved in ABA-induced antioxidant defence and functions upstream of ZmMPK5 in ABA signalling in maize. PMID:23268839

  8. 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.

  9. 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

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

    2016-10-01

    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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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 Central

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

    1997-01-01

    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. PMID:9390426

  15. Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene

    PubMed Central

    Timón, M; Beverley, P C L

    2001-01-01

    Expression of the leucocyte common antigen (CD45) in mammals is restricted to the nucleated lineages of haematopoietic cells. It appears in early progenitors in the bone marrow and is expressed at the surface of these cells throughout their differentiation. However, at least in T cells, the pattern of expression switches between different isoforms during the successive stages of differentiation in the thymus and after activation in the periphery. In order to understand the mechanisms controlling the transcription of the human CD45 gene, 2·7 kbp of the 5′-flanking region were sequenced and analysed for their ability to direct expression of a reporter gene. The only region with promoter activity was localized within the first intron of the gene. This promoter shows no tissue specificity but could be enhanced by a heterologous enhancer. Mobility shift assays showed complex but specific protein binding. The sequence in this region lacks similarity with known promoters or initiators but is highly conserved in evolution. No transcription initiation could be detected within or downstream of this region, suggesting that this might be a new type of RNA polymerase II promoter able to drive transcription from an upstream sequence. An additional exon was also found upstream of exon 1. The two exons 1 (1a and 1b) are mutually exclusive and both are spliced to exon 2. This makes the structure of the 5′ region of the human CD45 gene identical to its mouse homologue. PMID:11260323

  16. 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

  17. Conditional Inactivation of Upstream Binding Factor Reveals Its Epigenetic Functions and the Existence of a Somatic Nucleolar Precursor Body

    PubMed Central

    Hamdane, Nourdine; Stefanovsky, Victor Y.; Tremblay, Michel G.; Németh, Attila; Paquet, Eric; Lessard, Frédéric; Sanij, Elaine; Hannan, Ross; Moss, Tom

    2014-01-01

    Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs). PMID:25121932

  18. Combinatorial protein reagents to manipulate protein function.

    PubMed

    Colas, P

    2000-02-01

    The design and use of combinatorial protein libraries has become a fast moving field in molecular biology. Different experimental systems supporting various selection schemes are now available. The latest breakthroughs include evolutionary experiments to improve existing binding surfaces, selections of homodimerizing peptides, the use of peptide aptamers to disrupt protein interactions inside living cells, and functional selections of aptamers to probe regulatory networks.

  19. Arabidopsis Histidine Kinase CKI1 Acts Upstream of HISTIDINE PHOSPHOTRANSFER PROTEINS to Regulate Female Gametophyte Development and Vegetative Growth[C][W

    PubMed Central

    Deng, Yan; Dong, Haili; Mu, Jinye; Ren, Bo; Zheng, Binglian; Ji, Zhendong; Yang, Wei-Cai; Liang, Yan; Zuo, Jianru

    2010-01-01

    Cytokinin signaling is mediated by a multiple-step phosphorelay. Key components of the phosphorelay consist of the histidine kinase (HK)-type receptors, histidine phosphotransfer proteins (HP), and response regulators (RRs). Whereas overexpression of a nonreceptor-type HK gene CYTOKININ-INDEPENDENT1 (CKI1) activates cytokinin signaling by an unknown mechanism, mutations in CKI1 cause female gametophytic lethality. However, the function of CKI1 in cytokinin signaling remains unclear. Here, we characterize a mutant allele, cki1-8, that can be transmitted through female gametophytes with low frequency (∼0.17%). We have recovered viable homozygous cki1-8 mutant plants that grow larger than wild-type plants, show defective megagametogenesis and rarely set enlarged seeds. We found that CKI1 acts upstream of AHP (Arabidopsis HP) genes, independently of cytokinin receptor genes. Consistently, an ahp1,2-2,3,4,5 quintuple mutant, which contains an ahp2-2 null mutant allele, exhibits severe defects in megagametogenesis, with a transmission efficiency of <3.45% through female gametophytes. Rarely recovered ahp1,2-2,3,4,5 quintuple mutants are seedling lethal. Finally, the female gametophytic lethal phenotype of cki1-5 (a null mutant) can be partially rescued by IPT8 or ARR1 (a type-B Arabidopsis RR) driven by a CKI1 promoter. These results define a genetic pathway consisting of CKI1, AHPs, and type-B ARRs in the regulation of female gametophyte development and vegetative growth. PMID:20363773

  20. 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

  1. Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)*

    PubMed Central

    Shimamoto, Seiko; Kubota, Yasuo; Yamaguchi, Fuminori; Tokumitsu, Hiroshi; Kobayashi, Ryoji

    2013-01-01

    The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway. PMID:23344957

  2. [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.

  3. A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

    PubMed Central

    Naumann, Anja; Hochstein, Norbert; Weber, Stefanie; Fanning, Ellen; Doerfler, Walter

    2009-01-01

    We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins. PMID:19853235

  4. A distinct DNA-methylation boundary in the 5'- upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome.

    PubMed

    Naumann, Anja; Hochstein, Norbert; Weber, Stefanie; Fanning, Ellen; Doerfler, Walter

    2009-11-01

    We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.

  5. Upstream Regulatory Region Alterations Found in Human Papillomavirus Type 16 (HPV-16) Isolates from Cervical Carcinomas Increase Transcription, ori Function, and HPV Immortalization Capacity in Culture▿

    PubMed Central

    Lace, Michael J.; Isacson, Christina; Anson, James R.; Lörincz, Attila T.; Wilczynski, Sharon P.; Haugen, Thomas H.; Turek, Lubomír P.

    2009-01-01

    Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis. PMID:19458011

  6. Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice.

    PubMed

    Thépot, D; Devinoy, E; Fontaine, M L; Stinnakre, M G; Massoud, M; Kann, G; Houdebine, L M

    1995-11-01

    Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was radioimmunoassay into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0-16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

    2016-05-01

    The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4-DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3-DNA complex.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. JNK3 Enzyme Binding to Arrestin-3 Differentially Affects the Recruitment of Upstream Mitogen-activated Protein (MAP) Kinase Kinases*

    PubMed Central

    Zhan, Xuanzhi; Kaoud, Tamer S.; Kook, Seunghyi; Dalby, Kevin N.; Gurevich, Vsevolod V.

    2013-01-01

    Arrestin-3 was previously shown to bind JNK3α2, MKK4, and ASK1. However, full JNK3α2 activation requires phosphorylation by both MKK4 and MKK7. Using purified proteins we show that arrestin-3 directly interacts with MKK7 and promotes JNK3α2 phosphorylation by both MKK4 and MKK7 in vitro as well as in intact cells. The binding of JNK3α2 promotes an arrestin-3 interaction with MKK4 while reducing its binding to MKK7. Interestingly, the arrestin-3 concentration optimal for scaffolding the MKK7-JNK3α2 module is ∼10-fold higher than for the MKK4-JNK3α2 module. The data provide a mechanistic basis for arrestin-3-dependent activation of JNK3α2. The opposite effects of JNK3α2 on arrestin-3 interactions with MKK4 and MKK7 is the first demonstration that the kinase components in mammalian MAPK cascades regulate each other's interactions with a scaffold protein. The results show how signaling outcomes can be affected by the relative expression of scaffolding proteins and components of signaling cascades that they assemble. PMID:23960075

  18. Nitric Oxide Functions as a Signal and Acts Upstream of AtCaM3 in Thermotolerance in Arabidopsis Seedlings1[C][W

    PubMed Central

    Xuan, Yi; Zhou, Shuo; Wang, Lei; Cheng, Yudou; Zhao, Liqun

    2010-01-01

    To characterize the role of nitric oxide (NO) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on three types of Arabidopsis seedlings: wild type, noa1(rif1) (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) and nia1nia2 (for nitrate reductase [NR]-defective double mutant), which both exhibit reduced endogenous NO levels, and a rescued line of noa1(rif1). After HS treatment, the survival ratios of the mutant seedlings were lower than those of wild type; however, they were partially restored in the rescued line. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine revealed that internal NO affects heat sensitivity in a concentration-dependent manner. Calmodulin 3 (CaM3) is a key component of HS signaling in Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis after HS treatment revealed that the AtCaM3 mRNA level was regulated by the internal NO level. Sodium nitroprusside enhanced the survival of the wild-type and noa1(rif1) seedlings; however, no obvious effects were observed for cam3 single or cam3noa1(rif1) double mutant seedlings, suggesting that AtCaM3 is involved in NO signal transduction as a downstream factor. This point was verified by phenotypic analysis and thermotolerance testing using seedlings of three AtCaM3-overexpressing transgenic lines in an noa1(rif1) background. Electrophoretic mobility-shift and western-blot analyses demonstrated that after HS treatment, NO stimulated the DNA-binding activity of HS transcription factors and the accumulation of heat shock protein 18.2 (HSP18.2) through AtCaM3. These data indicate that NO functions in signaling and acts upstream of AtCaM3 in thermotolerance, which is dependent on increased HS transcription factor DNA-binding activity and HSP accumulation. PMID:20576787

  19. 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.

  20. A functional SNP upstream of the beta-2 adrenergic receptor gene (ADRB2) is associated with obesity in Oceanic populations.

    PubMed

    Naka, I; Hikami, K; Nakayama, K; Koga, M; Nishida, N; Kimura, R; Furusawa, T; Natsuhara, K; Yamauchi, T; Nakazawa, M; Ataka, Y; Ishida, T; Inaoka, T; Iwamoto, S; Matsumura, Y; Ohtsuka, R; Tsuchiya, N; Ohashi, J

    2013-09-01

    Obesity is a growing health concern in the Oceanic populations. To investigate the genetic factors associated with adult obesity in the Oceanic populations, the association of single nucleotide polymorphisms (SNPs) of the beta-2 adrenergic receptor (ADRB2) gene with obesity was examined in 694 adults living in Tonga and Solomon Islands. A screening for variation in 16 Oceanic subjects detected 17 SNPs in the entire region of ADRB2, of which nine SNPs including two non-synonymous ones, rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu), were further genotyped for all subjects. The rs34623097-A allele, at a SNP located upstream of ADRB2, showed the strongest association with risk for obesity in a logistic regression analysis adjusted for age, sex, and population (P=5.6 × 10(-4), odds ratio [OR]=2.5, 95% confidence interval [CI]=1.5-4.2). The 27Glu was also significantly associated with obesity in the single-point association analysis (P=0.013, OR=2.0, 95%CI=1.2-3.4); however, this association was no longer significant after adjustment for rs34623097 since these SNPs were in linkage disequilibrium with each other. A copy of the obesity-risk allele, rs34623097-A, led to a 1.6 kg/m(2) increase in body mass index (BMI; defined as weight in kilograms divided by height in meters squared) (P=0.0019). A luciferase reporter assay indicated that rs34623097-A reduced the transcriptional activity of the luciferase reporter gene by approximately 10% compared with rs34623097-G. An electrophoretic mobility shift assay demonstrated that rs34623097 modulated the binding affinity with nuclear factors. An evolutionary analysis implies that a G>A mutation at rs34623097 occurred in the Neandertal genome and then the rs34623097-A allele flowed into the ancestors of present-day humans. The present results suggest that rs34623097-A, which would lead to lower expression of ADRB2, contributes to the onset of obesity in the Oceanic populations.

  1. A functional SNP upstream of the beta-2 adrenergic receptor gene (ADRB2) is associated with obesity in Oceanic populations

    PubMed Central

    Naka, I; Hikami, K; Nakayama, K; Koga, M; Nishida, N; Kimura, R; Furusawa, T; Natsuhara, K; Yamauchi, T; Nakazawa, M; Ataka, Y; Ishida, T; Inaoka, T; Iwamoto, S; Matsumura, Y; Ohtsuka, R; Tsuchiya, N; Ohashi, J

    2013-01-01

    OBJECTIVE: Obesity is a growing health concern in the Oceanic populations. To investigate the genetic factors associated with adult obesity in the Oceanic populations, the association of single nucleotide polymorphisms (SNPs) of the beta-2 adrenergic receptor (ADRB2) gene with obesity was examined in 694 adults living in Tonga and Solomon Islands. RESULTS: A screening for variation in 16 Oceanic subjects detected 17 SNPs in the entire region of ADRB2, of which nine SNPs including two non-synonymous ones, rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu), were further genotyped for all subjects. The rs34623097-A allele, at a SNP located upstream of ADRB2, showed the strongest association with risk for obesity in a logistic regression analysis adjusted for age, sex, and population (P=5.6 × 10−4, odds ratio [OR]=2.5, 95% confidence interval [CI]=1.5–4.2). The 27Glu was also significantly associated with obesity in the single-point association analysis (P=0.013, OR=2.0, 95%CI=1.2–3.4); however, this association was no longer significant after adjustment for rs34623097 since these SNPs were in linkage disequilibrium with each other. A copy of the obesity-risk allele, rs34623097-A, led to a 1.6 kg/m2 increase in body mass index (BMI; defined as weight in kilograms divided by height in meters squared) (P=0.0019). A luciferase reporter assay indicated that rs34623097-A reduced the transcriptional activity of the luciferase reporter gene by approximately 10% compared with rs34623097-G. An electrophoretic mobility shift assay demonstrated that rs34623097 modulated the binding affinity with nuclear factors. An evolutionary analysis implies that a G>A mutation at rs34623097 occurred in the Neandertal genome and then the rs34623097-A allele flowed into the ancestors of present-day humans. CONCLUSION: The present results suggest that rs34623097-A, which would lead to lower expression of ADRB2, contributes to the onset of obesity in the Oceanic populations. PMID:23229733

  2. 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.

  3. 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.

  4. 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.

  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. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. The upstream regulatory region of the human papilloma virus-16 contains an E2 protein-independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones.

    PubMed Central

    Gloss, B; Bernard, H U; Seedorf, K; Klock, G

    1987-01-01

    The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters. Images Fig. 2. Fig. 6. PMID:2828035

  13. 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.

  14. SAP155-mediated c-myc suppressor far-upstream element-binding protein-interacting repressor splicing variants are activated in colon cancer tissues.

    PubMed

    Kajiwara, Toshiko; Matsushita, Kazuyuki; Itoga, Sakae; Tamura, Mai; Tanaka, Nobuko; Tomonaga, Takeshi; Matsubara, Hisahiro; Shimada, Hideaki; Habara, Yasuaki; Matsuo, Masafumi; Nomura, Fumio

    2013-02-01

    The c-myc transcriptional suppressor, far-upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), is alternatively spliced in colorectal cancer tissue (Matsushita et al., Cancer Res 2006). Recently, the knockdown of SAP155 pre-mRNA-splicing factor, a subunit of SF3b, was reported to disturb FIR pre-mRNA splicing and yield FIRΔexon2, an exon 2-spliced variant of FIR, which lacks c-myc repression activity. In the present study, novel splicing variants of FIR, Δ3 and Δ4, were also generated by SAP155 siRNA, and these variants were found to be activated in human colorectal cancer tissue. Furthermore, the expression levels of FIR variant mRNA were examined in the peripheral blood of colorectal cancer patients and healthy volunteers to assess its potency for tumor detection. As expected, circulating FIR variant mRNA in the peripheral blood of cancer patients were significantly overexpressed compared to that in healthy volunteers. In particular, the area under the receiving operating characteristic curve of FIR, FIRΔexon2 or FIRΔexon2/FIR, was greater than those of conventional carcinoembryonic antigen or carbohydrate antigen 19-9. In addition, FIRΔexon2 or FIR mRNA expression in the peripheral blood was significantly reduced after operative removal of colorectal tumors. Thus, circulating FIR and FIRΔexon2 mRNA are potential novel screening markers for colorectal cancer testing with conventional carcinoembryonic antigen and or carbohydrate antigen 19-9. Taken together, our results indicate that overexpression of FIR and its splicing variants in colorectal cancer directs feed-forward or addicted circuit c-myc transcriptional activation. Clinical implications for colorectal cancers of novel FIR splicing variants are also discussed in the present paper. © 2012 Japanese Cancer Association.

  15. 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

  16. 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.

  17. 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."

  18. 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

  19. Functional Foods Containing Whey Proteins

    USDA-ARS?s Scientific Manuscript database

    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...

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression.

    PubMed Central

    Einerhand, A W; Kos, W; Smart, W C; Kal, A J; Tabak, H F; Cooper, T G

    1995-01-01

    Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80. PMID:7760837

  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. 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.

  14. Characterization and Functionality of Corn Germ Proteins

    USDA-ARS?s Scientific Manuscript database

    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. The curved DNA structure in the 5′-upstream region of the light-responsive genes: its universality, binding factor and function for cyanobacterial psbA transcription

    PubMed Central

    Asayama, Munehiko; Kato, Hideki; Shibato, Junko; Shirai, Makoto; Ohyama, Takashi

    2002-01-01

    A unique DNA curvature, the CIT, has been found in the 5′-upstream region of the psbA2 gene, which exhibits basal, light-responsive and circadian rhythmic transcription, in a unicellular photosynthetic cyanobacterium, Microcystis aeruginosa K-81. In this study, we report the universality of curvatures found in 5′-upstream regions in the psbA family and the function of the curvature in gene expression. Intrinsic curvatures were identified within 1000 bp upstream from the psbA genes in another cyanobacterium, a red alga and in plants (monocot and dicot). Mutagenized curvatures were constructed and confirmed to have disrupted architecture by gel electrophoresis and atomic force microscopy. Relatively small amounts but light-responsive transcripts of psbA2 were observed in cyanobacterial transformants harboring the mutagenized curvature under light/dark and light/high-light conditions. This shows that the curvature is important for basal transcription. In vitro primer extension and DNA mobility shift assay revealed that factors which might bind to the region upstream from the bending center contribute to the effective basal transcription of psbA2. PMID:12409456

  17. 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.

  18. 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.

  19. 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.

  20. Concomitant expression of far upstream element (FUSE) binding protein (FBP) interacting repressor (FIR) and its splice variants induce migration and invasion of non-small cell lung cancer (NSCLC) cells.

    PubMed

    Müller, Benedikt; Bovet, Michael; Yin, Yi; Stichel, Damian; Malz, Mona; González-Vallinas, Margarita; Middleton, Alistair; Ehemann, Volker; Schmitt, Jennifer; Muley, Thomas; Meister, Michael; Herpel, Esther; Singer, Stephan; Warth, Arne; Schirmacher, Peter; Drasdo, Dirk; Matthäus, Franziska; Breuhahn, Kai

    2015-11-01

    Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. Sucrose Synthase: Expanding Protein Function

    USDA-ARS?s Scientific Manuscript database

    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...

  2. 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.

  3. 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

  4. S100 Proteins Modulate Protein Phosphatase 5 Function

    PubMed Central

    Yamaguchi, Fuminori; Umeda, Yoshinori; Shimamoto, Seiko; Tsuchiya, Mitsumasa; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2012-01-01

    PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca2+/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca2+-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction. PMID:22399290

  5. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    PubMed Central

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  6. Graph pyramids for protein function prediction

    PubMed Central

    2015-01-01

    Background Uncovering the hidden organizational characteristics and regularities among biological sequences is the key issue for detailed understanding of an underlying biological phenomenon. Thus pattern recognition from nucleic acid sequences is an important affair for protein function prediction. As proteins from the same family exhibit similar characteristics, homology based approaches predict protein functions via protein classification. But conventional classification approaches mostly rely on the global features by considering only strong protein similarity matches. This leads to significant loss of prediction accuracy. Methods Here we construct the Protein-Protein Similarity (PPS) network, which captures the subtle properties of protein families. The proposed method considers the local as well as the global features, by examining the interactions among 'weakly interacting proteins' in the PPS network and by using hierarchical graph analysis via the graph pyramid. Different underlying properties of the protein families are uncovered by operating the proposed graph based features at various pyramid levels. Results Experimental results on benchmark data sets show that the proposed hierarchical voting algorithm using graph pyramid helps to improve computational efficiency as well the protein classification accuracy. Quantitatively, among 14,086 test sequences, on an average the proposed method misclassified only 21.1 sequences whereas baseline BLAST score based global feature matching method misclassified 362.9 sequences. With each correctly classified test sequence, the fast incremental learning ability of the proposed method further enhances the training model. Thus it has achieved more than 96% protein classification accuracy using only 20% per class training data. PMID:26044522

  7. Graph pyramids for protein function prediction.

    PubMed

    Sandhan, Tushar; Yoo, Youngjun; Choi, Jin; Kim, Sun

    2015-01-01

    Uncovering the hidden organizational characteristics and regularities among biological sequences is the key issue for detailed understanding of an underlying biological phenomenon. Thus pattern recognition from nucleic acid sequences is an important affair for protein function prediction. As proteins from the same family exhibit similar characteristics, homology based approaches predict protein functions via protein classification. But conventional classification approaches mostly rely on the global features by considering only strong protein similarity matches. This leads to significant loss of prediction accuracy. Here we construct the Protein-Protein Similarity (PPS) network, which captures the subtle properties of protein families. The proposed method considers the local as well as the global features, by examining the interactions among 'weakly interacting proteins' in the PPS network and by using hierarchical graph analysis via the graph pyramid. Different underlying properties of the protein families are uncovered by operating the proposed graph based features at various pyramid levels. Experimental results on benchmark data sets show that the proposed hierarchical voting algorithm using graph pyramid helps to improve computational efficiency as well the protein classification accuracy. Quantitatively, among 14,086 test sequences, on an average the proposed method misclassified only 21.1 sequences whereas baseline BLAST score based global feature matching method misclassified 362.9 sequences. With each correctly classified test sequence, the fast incremental learning ability of the proposed method further enhances the training model. Thus it has achieved more than 96% protein classification accuracy using only 20% per class training data.

  8. 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.

  9. 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.

  10. 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.

  11. Towards site-based protein functional annotations.

    PubMed

    Lei, Seak Fei; Huan, Jun

    2010-01-01

    The exact relationship between protein active centres and protein functions is unclear even after decades of intensive study. To improve functional prediction ability based on the local structures, we proposed three different methods. 1. We used Markov Random Field (MRF) to describe protein active region. 2. We developed filtering method that considers the local environment around the active sites. 3. We created multiple structure motifs by extending the motif to neighbouring residues. Our experiment results with enzyme families < 40% sequence identity demonstrated that our methods reduced random matches and could improve up to 70% of the functional annotation ability (using area under curve).

  12. Cyclophilins: proteins in search of function.

    PubMed

    Kumari, Sumita; Roy, Suchismita; Singh, Prabhjeet; Singla-Pareek, Sneh L; Pareek, Ashwani

    2013-01-01

    Cyclophilins constitute a subgroup of large family of proteins called immunophilins, which also include FKBPs and Parvulins. They are remarkably conserved in all genera, highlighting their pivotal role in important cellular processes. Most cyclophilins display PPIase enzymatic activity, multiplicity, diverse cellular locations and active role in protein folding which render them to be included in the class of diverse set of proteins called molecular chaperones. Due to their distinct PPIase function, besides protein disulfide isomerases and protein foldases, cyclophilins have been deemed necessary for in vivo chaperoning activity. Unlike other cellular chaperones, these proteins are specific in their respective targets. Not all cyclophilin proteins possess PPIase activity, indicating a loss of their PPIase activity during the course of evolution and gain of function independent of their PPIase activity. The PPIase function of cyclophilins is also compensated by their functional homologs, like FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have been reported to be associated with diverse functions and regulatory pathways through their foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins were reported or suggested, the physiological relevance and molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. However, their wide distribution and ubiquitous nature signifies their fundamental importance in plant survival. Several of these members have also been directly linked to multiple stresses. This review attempts to deal with plant cyclophilins with respect to their role in stress response.

  13. 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.

  14. 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.

  15. 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.

  16. Integrated protein function prediction by mining function associations, sequences, and protein-protein and gene-gene interaction networks.

    PubMed

    Cao, Renzhi; Cheng, Jianlin

    2016-01-15

    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. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. 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

  18. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  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. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. Nanoscale hybrid protein/polymer functionalized materials

    NASA Astrophysics Data System (ADS)

    Ho, Dean; Chu, Ben; Lee, Hyeseung; Montemagno, Carlo D.

    2004-07-01

    Block copolymer-based membrane technology represents a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. Our work has demonstrated the fabrication of large-area, protein- enhanced membranes that possess significant performance improvements in protein functionality. Among its many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods like Langmuir-Blodgett (LB) deposition, or spontaneous protein-functionalized nano-vesicle formation. The membrane protein, Bacteriorhodopsin (BR), found in Halobacterium Halobium, is a light-actuated proton pump that develops gradients towards the demonstration of coupled functionality with other membrane proteins to effect ATP production, or production of electricity through Bacteriorhodopsin activity-dependent reversal of Cytochrome C Oxidase (COX), found in Rhodobacter Sphaeroides. Using quantum dot-labeled, engineered protein constructs, we have demonstrated large-scale insertion of proteins into block copolymer Langmuir-Blodgett (LB) films as well as measurable pH changes based upon light-actuated proton pumping. Light actuated-activity across the protein-functionalized membrane when fully enclosed in a sol-gel matrix has also been observed using impedance spectroscopy. Initial data has suggested a significant pH change of up to 1.75 in a volume of 100 mL and surface area of 0.317cm2, a level that is capable of powering a number of proton-gradient dependent proteins towards the buildup of a robust, hybrid protein/polymer device. Recent atomic force microscopy studies of the protein-embedded polymer film samples have revealed the formation of protein aggregate-based pattern generation with very uniform torus-shaped rings. Current work focused towards characterizing the effects that various pattern formations can have on the

  7. Evolution of functionality in lattice proteins

    PubMed Central

    Williams, Paul D.; Pollock, David D.; Goldstein, Richard A.

    2010-01-01

    We study the evolution of protein functionality using a two-dimensional lattice model. The characteristics particular to evolution, such as population dynamics and early evolutionary trajectories, have a large effect on the distribution of observed structures. Only subtle differences are observed between the distribution of structures evolved for function and those evolved for their ability to form compact structures. PMID:11381526

  8. 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.

  9. Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

    PubMed

    Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

    2016-11-15

    Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Functions of Intracellular Retinoid Binding-Proteins

    PubMed Central

    2017-01-01

    Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function. PMID:27830500

  16. S100A6 protein: functional roles.

    PubMed

    Donato, Rosario; Sorci, Guglielmo; Giambanco, Ileana

    2017-08-01

    S100A6 protein belongs to the A group of the S100 protein family of Ca(2+)-binding proteins. It is expressed in a limited number of cell types in adult normal tissues and in several tumor cell types. As an intracellular protein, S100A6 has been implicated in the regulation of several cellular functions, such as proliferation, apoptosis, the cytoskeleton dynamics, and the cellular response to different stress factors. S100A6 can be secreted/released by certain cell types which points to extracellular effects of the protein. RAGE (receptor for advanced glycation endproducts) and integrin β1 transduce some extracellular S100A6's effects. Dosage of serum S100A6 might aid in diagnosis in oncology.

  17. 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

  18. Regulation of protein function by native metastability

    PubMed Central

    Lee, Cheolju; Park, Soon-Ho; Lee, Min-Youn; Yu, Myeong-Hee

    2000-01-01

    In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of α1-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions. PMID:10884404

  19. 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.

  20. Functional Analyses of Vertebrate TCF Proteins in C. elegans Embryos

    PubMed Central

    Robertson, Scott M.; Lo, Miao-Chia; Odom, Ranaan; Yang, Xiao-Dong; Medina, Jessica; Huang, Shuyi; Lin, Rueyling

    2011-01-01

    In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognize specific promoter sequences upstream of endoderm genes and interact with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and

  1. Evolution-Based Functional Decomposition of Proteins

    PubMed Central

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

    2016-01-01

    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. PMID:27254668

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Hierarchical ensemble methods for protein function prediction.

    PubMed

    Valentini, Giorgio

    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.

  9. 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.

  10. FUNCTIONALITY OF MEMBRANE SEPARATED EGG WHITE PROTEINS

    USDA-ARS?s Scientific Manuscript database

    The excellent nutritional and functional properties of liquid egg white (LEW), which is essentially a viscous fat-free protein solution, are exploited in many food preparations. Thermal pasteurization (at 56.6oC for 3.5 min. minimum) is currently used by industry to eliminate the microflora in LEW ...

  11. Modification of sorghum proteins for enhanced functionality

    USDA-ARS?s Scientific Manuscript database

    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...

  12. 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.

  13. Functional roles of protein splicing factors

    PubMed Central

    Chen, Hsin-Chou; Cheng, Soo-Chen

    2012-01-01

    RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system. PMID:22762203

  14. Signaling Function of Heme Oxygenase Proteins

    PubMed Central

    2014-01-01

    Abstract Significance: Many reports have underscored the importance of the heme degradation pathway that is regulated by heme oxygenase (HO). This reaction releases bile pigments and carbon monoxide (CO), which are important antioxidant and signaling molecules. Thus, the reaction of HO-1 would have significant cytoprotective effects. Nevertheless, the importance of this protein goes beyond its enzymatic action. New evidence outlines significant effects of inactive forms of the HO-1 protein. Recent Advances: In fact, the role of the HO protein in cellular signaling, including transcription factor activation, binding to proteins, phosphorylation, and modulation of protein function, among others, has started being elucidated. The mechanism by which the inducible form of HO-1, in particular, can migrate to various cellular compartments to mediate important signaling or how and why it binds to key transcription factors and other proteins that are important in DNA repair is also described in several physiologic systems. Critical Issues: The signaling functions of HO-1 may have particular relevance in clinical circumstances, including cancer, as redistribution of HO-1 into the nuclear compartment is observed with cancer progression and metastasis. In addition, along with oxidative stress, the pleiotropic functions of HO-1 modulate antioxidant defense. In organ transplantation, HO and its byproducts suppress rejection at multiple levels and in sepsis-induced pulmonary dysfunction, inhaled CO or modulation of HO activity can change the course of the disease in animals. Future Directions: It is hoped that a more detailed understanding of the various signaling functions of HO will guide therapeutic approaches for complex diseases. Antioxid. Redox Signal. 20, 1743–1753. PMID:24180238

  15. Ion distributions upstream of an interplanetary shock

    NASA Astrophysics Data System (ADS)

    Kajdic, Primoz; Hietala, Heli; Blanco-Cano, Xochitl

    2017-04-01

    It is well known that supercritical collisionless shocks in the interplanetary (IP) space reflect part of the incoming particles (ions, electrons) in order to dissipate the kinetic energy of the upstream solar wind flow. When the conditions are right the reflected particles can escape far upstream from the shock. Their interaction with incoming ions and electrons results in the formation of the foreshock region which is populated by ultra-low frequency magnetic field fluctuations and different populations of reflected ions. Our knowledge on the latter comes mostly from observations of our planet's foreshock. However, the bow shock of the Earth typically has high Mach numbers, and the relatively small global curvature radius of the shock's shape affects the ion distribution characteristics. Interplanetary (IP) shocks, on the other hand, typically have lower Mach numbers and larger global curvature radii. In the past the majority of observed ion distributions detected upstream of IP shocks were diffuse. In only a couple of works the field-aligned ion beams were reported and even then the details of the ion distributions functions could not be determined. Here we present the first study showing clear observations of different types of ion distributions upstream of an interplanetary shock. The shock was observed on 8 October 2013 by several spacecraft, namely Wind, ACE, and the two ARTEMIS spacecraft P1 and P2. By using combined data from the Electrostatic Analyzer and the Solid State Telescope instruments onboard both ARTEMIS spacecraft we observed different types of ion distributions upstream of the shock: The distributions changed from field-aligned ion beams that were detected farthest from the shock, to intermediate and then to almost diffuse ion distributions near the shock transition. Furthermore, the observations at P1 and P2 locations also show spatial variability of the foreshock and the IP shock. The angle between the local shock normal and the upstream

  16. Arabidopsis OST1 Protein Kinase Mediates the Regulation of Stomatal Aperture by Abscisic Acid and Acts Upstream of Reactive Oxygen Species Production

    PubMed Central

    Mustilli, Anna-Chiara; Merlot, Sylvain; Vavasseur, Alain; Fenzi, Francesca; Giraudat, Jérôme

    2002-01-01

    During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO2, suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H2O2 or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed. PMID:12468729

  17. 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

  18. The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function

    PubMed Central

    van Nocker, Steven; Ludwig, Philip

    2003-01-01

    Background The WD motif (also known as the Trp-Asp or WD40 motif) is found in a multitude of eukaryotic proteins involved in a variety of cellular processes. Where studied, repeated WD motifs act as a site for protein-protein interaction, and proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins. In the model plant Arabidopsis thaliana, members of this superfamily are increasingly being recognized as key regulators of plant-specific developmental events. Results We analyzed the predicted complement of WDR proteins from Arabidopsis, and compared this to those from budding yeast, fruit fly and human to illustrate both conservation and divergence in structure and function. This analysis identified 237 potential Arabidopsis proteins containing four or more recognizable copies of the motif. These were classified into 143 distinct families, 49 of which contained more than one Arabidopsis member. Approximately 113 of these families or individual proteins showed clear homology with WDR proteins from the other eukaryotes analyzed. Where conservation was found, it often extended across all of these organisms, suggesting that many of these proteins are linked to basic cellular mechanisms. The functional characterization of conserved WDR proteins in Arabidopsis reveals that these proteins help adapt basic mechanisms for plant-specific processes. Conclusions Our results show that most Arabidopsis WDR proteins are strongly conserved across eukaryotes, including those that have been found to play key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and /or structure outside of the WDR regions. PMID:14672542

  19. 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.

  20. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

    Avila, Frank W.; Sirot, Laura K.; LaFlamme, Brooke A.; Rubinstein, C. Dustin; Wolfner, Mariana F.

    2014-01-01

    Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects. PMID:20868282

  1. Ice-Binding Proteins and Their Function.

    PubMed

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-02

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.

  2. 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.

  3. 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.

  4. 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.

  5. Functional peptides derived from rice bran proteins.

    PubMed

    Liu, Y Q; Strappe, P; Shang, W T; Zhou, Z K

    2017-09-08

    Rice bran has been predominantly used in the feed industry, and only recently it has attracted greater attention in terms of human nutrition with increasing knowledge of its bioactivity. A growing interest is the analysis of physiologically active peptides derived from rice bran proteins. In this paper, the bioactivities of rice bran proteins hydrolysates and peptides are reviewed based on recent studies. These enzymatic hydrolysates and peptides exert various biological activities including antioxidant, antidiabetic, anticancer and inhibitory activity for angiotensin converting enzyme (ACE), which may ultimately prevent certain chronic diseases. Nevertheless, these functionalities can be highly associated with their corresponding structural characteristics, in particular specific sequences and molecular weight distribution. This article may facilitate the expansion of the prospective applications of the bioactive peptides in a number of fields and provide some clues of the relationship between peptides structure and functionality for future research.

  6. 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

  7. 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.

  8. Upstream reciprocity in heterogeneous networks.

    PubMed

    Iwagami, Akio; Masuda, Naoki

    2010-08-07

    Many mechanisms for the emergence and maintenance of altruistic behavior in social dilemma situations have been proposed. Indirect reciprocity is one such mechanism, where other-regarding actions of a player are eventually rewarded by other players with whom the original player has not interacted. The upstream reciprocity (also called generalized indirect reciprocity) is a type of indirect reciprocity and represents the concept that those helped by somebody will help other unspecified players. In spite of the evidence for the enhancement of helping behavior by upstream reciprocity in rats and humans, theoretical support for this mechanism is not strong. In the present study, we numerically investigate upstream reciprocity in heterogeneous contact networks, in which the players generally have different number of neighbors. We show that heterogeneous networks considerably enhance cooperation in a game of upstream reciprocity. In heterogeneous networks, the most generous strategy, by which a player helps a neighbor on being helped and in addition initiates helping behavior, first occupies hubs in a network and then disseminates to other players. The scenario to achieve enhanced altruism resembles that seen in the case of the Prisoner's Dilemma game in heterogeneous networks. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. Isolation of a Novel Family of C2H2 Zinc Finger Proteins Implicated in Transcriptional Repression Mediated by Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) Orphan Nuclear Receptors*

    PubMed Central

    Avram, Dorina; Fields, Andrew; Top, Karen Pretty On; Nevrivy, Daniel J.; Ishmael, Jane E.; Leid, Mark

    2010-01-01

    Two novel and related C2H2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors. PMID:10744719

  10. 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.

  11. 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

  12. Improving the prediction of yeast protein function using weighted protein-protein interactions

    PubMed Central

    2011-01-01

    Background Bioinformatics can be used to predict protein function, leading to an understanding of cellular activities, and equally-weighted protein-protein interactions (PPI) are normally used to predict such protein functions. The present study provides a weighting strategy for PPI to improve the prediction of protein functions. The weights are dependent on the local and global network topologies and the number of experimental verification methods. The proposed methods were applied to the yeast proteome and integrated with the neighbour counting method to predict the functions of unknown proteins. Results A new technique to weight interactions in the yeast proteome is presented. The weights are related to the network topology (local and global) and the number of identified methods, and the results revealed improvement in the sensitivity and specificity of prediction in terms of cellular role and cellular locations. This method (new weights) was compared with a method that utilises interactions with the same weight and it was shown to be superior. Conclusions A new method for weighting the interactions in protein-protein interaction networks is presented. Experimental results concerning yeast proteins demonstrated that weighting interactions integrated with the neighbor counting method improved the sensitivity and specificity of prediction in terms of two functional categories: cellular role and cell locations. PMID:21524280

  13. 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.

  14. 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.

  15. 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.

  16. Plugging into proteins: poisoning protein function by a hydrophobic nanoparticle.

    PubMed

    Zuo, Guanghong; Huang, Qing; Wei, Guanghong; Zhou, Ruhong; Fang, Haiping

    2010-12-28

    Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the nanoparticle toxicity and other risks involved with these nanoparticles to human health. In this study, we use large-scale molecular dynamics simulations to study the interaction between several proteins (WW domains) and carbon nanotubes (one form of hydrophobic nanoparticles). We have found that the carbon nanotube can plug into the hydrophobic core of proteins to form stable complexes. This plugging of nanotubes disrupts and blocks the active sites of WW domains from binding to the corresponding ligands, thus leading to the loss of the original function of the proteins. The key to this observation is the hydrophobic interaction between the nanoparticle and the hydrophobic residues, particularly tryptophans, in the core of the domain. We believe that these findings might provide a novel route to the nanoparticle toxicity on the molecular level for the hydrophobic nanoparticles.

  17. Nanostructured functional films from engineered repeat proteins

    PubMed Central

    Grove, Tijana Z.; Regan, Lynne; Cortajarena, Aitziber L.

    2013-01-01

    Fundamental advances in biotechnology, medicine, environment, electronics and energy require methods for precise control of spatial organization at the nanoscale. Assemblies that rely on highly specific biomolecular interactions are an attractive approach to form materials that display novel and useful properties. Here, we report on assembly of films from the designed, rod-shaped, superhelical, consensus tetratricopeptide repeat protein (CTPR). We have designed three peptide-binding sites into the 18 repeat CTPR to allow for further specific and non-covalent functionalization of films through binding of fluorescein labelled peptides. The fluorescence signal from the peptide ligand bound to the protein in the solid film is anisotropic, demonstrating that CTPR films can impose order on otherwise isotropic moieties. Circular dichroism measurements show that the individual protein molecules retain their secondary structure in the film, and X-ray scattering, birefringence and atomic force microscopy experiments confirm macroscopic alignment of CTPR molecules within the film. This work opens the door to the generation of innovative biomaterials with tailored structure and function. PMID:23594813

  18. Xanthophylls as modulators of membrane protein function.

    PubMed

    Ruban, Alexander V; Johnson, Matthew P

    2010-12-01

    This review discusses the structural aspect of the role of photosynthetic antenna xanthophylls. It argues that xanthophyll hydrophobicity/polarity could explain the reason for xanthophyll variety and help to understand their recently emerging function--control of membrane organization and the work of membrane proteins. The structure of a xanthophyll molecule is discussed in relation to other amphiphilic compounds like lipids, detergents, etc. Xanthophyll composition of membrane proteins, the role of their variety in protein function are discussed using as an example for the major light harvesting antenna complex of photosystem II, LHCII, from higher plants. A new empirical parameter, hydrophobicity parameter (H-parameter), has been introduced as an effective measure of the hydrophobicity of the xanthophyll complement of LHCII from different xanthophyll biosynthesis mutants of Arabidopsis. Photosystem II quantum efficiency was found to correlate well with the H-parameter of LHCII xanthophylls. PSII down-regulation by non-photochemical chlorophyll fluorescence quenching, NPQ, had optimum corresponding to the wild-type xanthophyll composition, where lutein occupies intrinsic sites, L1 and L2. Xanthophyll polarity/hydrophobicity alteration by the activity of the xanthophyll cycle explains the allosteric character of NPQ regulation, memory of illumination history and the hysteretic nature of the relationship between the triggering factor, ΔpH, and the energy dissipation process. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. 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.

  20. Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species

    PubMed Central

    Wythe, Joshua D.; Liu, Jiandong; Cartry, Jerome; Vogler, Georg; Mohapatra, Bhagyalaxmi; Otway, Robyn T.; Huang, Yu; King, Isabelle N.; Maillet, Marjorie; Zheng, Yi; Crawley, Timothy; Taghli-Lamallem, Ouarda; Semsarian, Christopher; Dunwoodie, Sally; Winlaw, David; Harvey, Richard P.; Fatkin, Diane; Towbin, Jeffrey A.; Molkentin, Jeffery D.; Srivastava, Deepak; Ocorr, Karen; Bruneau, Benoit G.

    2011-01-01

    Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart–specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K+ channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42–Tinman interaction. To determine whether a Cdc42–Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1. PMID:21690310

  1. 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

  2. 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.

  3. 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).

  4. Physiological Functions of APP Family Proteins

    PubMed Central

    Müller, Ulrike C.; Zheng, Hui

    2012-01-01

    Biochemical and genetic evidence establishes a central role of the amyloid precursor protein (APP) in Alzheimer disease (AD) pathogenesis. Biochemically, deposition of the β-amyloid (Aβ) peptides produced from proteolytic processing of APP forms the defining pathological hallmark of AD; genetically, both point mutations and duplications of wild-type APP are linked to a subset of early onset of familial AD (FAD) and cerebral amyloid angiopathy. As such, the biological functions of APP and its processing products have been the subject of intense investigation, and the past 20+ years of research have met with both excitement and challenges. This article will review the current understanding of the physiological functions of APP in the context of APP family members. PMID:22355794

  5. 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

  6. 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.

  7. 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.

  8. Computer-aided design of functional protein interactions.

    PubMed

    Mandell, Daniel J; Kortemme, Tanja

    2009-11-01

    Predictive methods for the computational design of proteins search for amino acid sequences adopting desired structures that perform specific functions. Typically, design of 'function' is formulated as engineering new and altered binding activities into proteins. Progress in the design of functional protein-protein interactions is directed toward engineering proteins to precisely control biological processes by specifically recognizing desired interaction partners while avoiding competitors. The field is aiming for strategies to harness recent advances in high-resolution computational modeling-particularly those exploiting protein conformational variability-to engineer new functions and incorporate many functional requirements simultaneously.

  9. 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.

  10. 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.

  11. 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.

  12. Characterization of the functional properties of carob germ proteins

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Upstream waves in Saturn's foreshock

    NASA Technical Reports Server (NTRS)

    Bavassano Cattaneo, M. B.; Cattaneo, P.; Moreno, G.; Lepping, R. P.

    1991-01-01

    An analysis based on plasma and magnetic-field data obtained from Voyager 1 during its Saturn encounter is reported. The plasma data provided every 96 sec and magnetic-field data averaged over 48 sec are utilized. The evidence of upstream waves at Saturn are detected. The waves have a period, in the spacecraft frame, of about 550 sec and a relative amplitude larger than 0.3, are left- and right-hand elliptically polarized, and propagate at about 30 deg with respect to the average magnetic field. The appearance of the waves is correlated with the spacecraft being magnetically connected to the bow shock.

  14. 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.

  15. 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.

  16. 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

  17. Integrating protein-protein interactions and text mining for protein function prediction

    PubMed Central

    Jaeger, Samira; Gaudan, Sylvain; Leser, Ulf; Rebholz-Schuhmann, Dietrich

    2008-01-01

    Background Functional annotation of proteins remains a challenging task. Currently the scientific literature serves as the main source for yet uncurated functional annotations, but curation work is slow and expensive. Automatic techniques that support this work are still lacking reliability. We developed a method to identify conserved protein interaction graphs and to predict missing protein functions from orthologs in these graphs. To enhance the precision of the results, we furthermore implemented a procedure that validates all predictions based on findings reported in the literature. Results Using this procedure, more than 80% of the GO annotations for proteins with highly conserved orthologs that are available in UniProtKb/Swiss-Prot could be verified automatically. For a subset of proteins we predicted new GO annotations that were not available in UniProtKb/Swiss-Prot. All predictions were correct (100% precision) according to the verifications from a trained curator. Conclusion Our method of integrating CCSs and literature mining is thus a highly reliable approach to predict GO annotations for weakly characterized proteins with orthologs. PMID:18673526

  18. Protein species and moonlighting proteins: Very small changes in a protein's covalent structure can change its biochemical function.

    PubMed

    Jeffery, Constance J

    2016-02-16

    In the past few decades, hundreds of moonlighting proteins have been identified that perform two or more distinct and physiologically relevant biochemical or biophysical functions that are not due to gene fusions, multiple RNA splice variants, or pleiotropic effects. For this special issue on protein species, this article discusses three topics related to moonlighting proteins that illustrate how small changes or differences in protein covalent structures can result in different functions. Examples are given of moonlighting proteins that switch between functions after undergoing post-translational modifications (PTMs), proteins that share high levels of amino acid sequence identity to a moonlighting protein but share only one of its functions, and several "neomorphic moonlighting proteins" in which a single amino acid mutation results in the addition of a new function. For this special issue on protein species, this article discusses three topics related to moonlighting proteins : Post-translational modifications (PTMs) that can cause a switch between functions, homologs that share only one of multiple functions, and proteins in which a single amino acid mutation results in the creation of a new function. The examples included illustrate that even in an average protein of hundreds of amino acids, a relatively small difference in sequence or PTMs can result in a large difference in function, which can be important in predicting protein functions, regulation of protein functions, and in the evolution of new functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. 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.

  20. 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.

  1. Protein Function Prediction: Towards Integration of Similarity Metrics

    PubMed Central

    Erdin, Serkan; Lisewski, Andreas Martin; Lichtarge, Olivier

    2011-01-01

    Summary Genomics centers discover increasingly many protein sequences and structures, but not necessarily their full biological functions. Thus, currently, fewer than one percent of proteins have experimentally verified biochemical activities. To fill this gap, function prediction algorithms apply metrics of similarity between proteins on the premise that those sufficiently alike in sequence, or structure, will perform identical functions. Although high sensitivity is elusive, network analyses that integrate these metrics together hold the promise of rapid gains in function prediction specificity. PMID:21353529

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. INTEGRATING COMPUTATIONAL PROTEIN FUNCTION PREDICTION INTO DRUG DISCOVERY INITIATIVES

    PubMed Central

    Grant, Marianne A.

    2014-01-01

    Pharmaceutical researchers must evaluate vast numbers of protein sequences and formulate innovative strategies for identifying valid targets and discovering leads against them as a way of accelerating drug discovery. The ever increasing number and diversity of novel protein sequences identified by genomic sequencing projects and the success of worldwide structural genomics initiatives have spurred great interest and impetus in the development of methods for accurate, computationally empowered protein function prediction and active site identification. Previously, in the absence of direct experimental evidence, homology-based protein function annotation remained the gold-standard for in silico analysis and prediction of protein function. However, with the continued exponential expansion of sequence databases, this approach is not always applicable, as fewer query protein sequences demonstrate significant homology to protein gene products of known function. As a result, several non-homology based methods for protein function prediction that are based on sequence features, structure, evolution, biochemical and genetic knowledge have emerged. Herein, we review current bioinformatic programs and approaches for protein function prediction/annotation and discuss their integration into drug discovery initiatives. The development of such methods to annotate protein functional sites and their application to large protein functional families is crucial to successfully utilizing the vast amounts of genomic sequence information available to drug discovery and development processes. PMID:25530654

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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. Copyright © 2013 Wiley Periodicals, Inc.

  17. Protein mechanics: how force regulates molecular function.

    PubMed

    Seifert, Christian; Gräter, Frauke

    2013-10-01

    Regulation of proteins is ubiquitous and vital for any organism. Protein activity can be altered chemically, by covalent modifications or non-covalent binding of co-factors. Mechanical forces are emerging as an additional way of regulating proteins, by inducing a conformational change or by partial unfolding. We review some advances in experimental and theoretical techniques to study protein allostery driven by mechanical forces, as opposed to the more conventional ligand driven allostery. In this respect, we discuss recent single molecule pulling experiments as they have substantially augmented our view on the protein allostery by mechanical signals in recent years. Finally, we present a computational analysis technique, Force Distribution Analysis, that we developed to reveal allosteric pathways in proteins. Any kind of external perturbation, being it ligand binding or mechanical stretching, can be viewed as an external force acting on the macromolecule, rendering force-based experimental or computational techniques, a very general approach to the mechanics involved in protein allostery. This unifying view might aid to decipher how complex allosteric protein machineries are regulated on the single molecular level. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. 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.

  19. Dual function lipin proteins and glycerolipid metabolism.

    PubMed

    Harris, Thurl E; Finck, Brian N

    2011-06-01

    Lipin family proteins are emerging as crucial regulators of lipid metabolism. In triglyceride synthesis, lipins act as lipid phosphatase enzymes at the endoplasmic reticular membrane, catalyzing the dephosphorylation of phosphatidic acid to form diacylglycerol, which is the penultimate step in this process. However, lipin proteins are not integral membrane proteins, and can rapidly translocate within the cell. In fact, emerging evidence suggests that lipins also play crucial roles in the nucleus as transcriptional regulatory proteins. Thus, lipins are poised to regulate cellular lipid metabolism at multiple regulatory nodal points. This review summarizes the history of lipin proteins, and discusses the current state of our understanding of lipin biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. The physical characteristics of human proteins in different biological functions.

    PubMed

    Wang, Tengjiao; Tang, Hailin

    2017-01-01

    The physical properties of gene products are the foundation of their biological functions. In this study, we systematically explored relationships between physical properties and biological functions. The physical properties including origin time, evolution pressure, mRNA and protein stability, molecular weight, hydrophobicity, acidity/alkaline, amino acid compositions, and chromosome location. The biological functions are defined from 4 aspects: biological process, molecular function, cellular component and cell/tissue/organ expression. We found that the proteins associated with basic material and energy metabolism process originated earlier, while the proteins associated with immune, neurological system process etc. originated later. Tissues may have a strong influence on evolution pressure. The proteins associated with energy metabolism are double-stable. Immune and peripheral cell proteins tend to be mRNA stable/protein unstable. There are very few function items with double-unstable of mRNA and protein. The proteins involved in the cell adhesion tend to consist of large proteins with high proportion of small amino acids. The proteins of organic acid transport, neurological system process and amine transport have significantly high hydrophobicity. Interestingly, the proteins involved in olfactory receptor activity tend to have high frequency of aromatic, sulfuric and hydroxyl amino acids.

  9. Protein function annotation by homology-based inference

    PubMed Central

    Loewenstein, Yaniv; Raimondo, Domenico; Redfern, Oliver C; Watson, James; Frishman, Dmitrij; Linial, Michal; Orengo, Christine; Thornton, Janet; Tramontano, Anna

    2009-01-01

    With many genomes now sequenced, computational annotation methods to characterize genes and proteins from their sequence are increasingly important. The BioSapiens Network has developed tools to address all stages of this process, and here we review progress in the automated prediction of protein function based on protein sequence and structure. PMID:19226439

  10. [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.

  11. 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.

  12. 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

  13. 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.

  14. Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

    PubMed Central

    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.

    2014-01-01

    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. PMID:24429493

  15. Order of application determines the interaction between phorbol esters and GTP-gamma-S in dorsal raphe neurons: evidence that the effect of 5-HT is modified upstream of the G protein Ca channel interaction.

    PubMed

    Chen, Y; Penington, N J

    1997-05-01

    Phorbol esters activating protein kinase C (PKC) partially uncouple the inhibitory effect of serotonin (5-HT) from serotonergic neuron Ca2+ current. Presently the site of action of PKC is not known and may be the receptor, G protein, or ion channel. We recorded Ca2+ current from acutely isolated neurons with the use of the patch-clamp technique to study the site of action of PKC. Activation of the G protein with internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) occluded the response to 5-HT, but unexpectedly this effect was not reversed by the addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) despite the voltage-dependent reversal of the effect of GTP-gamma-S by long depolarizing steps to +80 mV. PMA was, however, able to partially reverse 5-HT-induced inhibition of Ca2+ current. The rate of reinhibition of the Ca2+ current (related to the concentration of activated G proteins) by GTP-gamma-S after the addition of PMA at -50 mV was identical to the rate when only GTP-gamma-S was present. By contrast, when cells were exposed first to PMA, and then GTP-gamma-S was perfused into the cell, GTP-gamma-S lost about half of its ability to activate the G protein. The rate of reinhibition of the Ca2+ current by internal GTP-gamma-S was also reduced in cells pretreated with PMA. The original result in which PMA did not reverse the action of GTP-gamma-S suggested that the channel was not the functional site of action of PMA, nor was the site on the G protein that binds to the channel, but it did not rule out the receptor. When the receptor was bypassed, after prior PKC activation, it was found that direct activation of the G protein by a nonhydrolyzable analogue of GTP was reduced; taken as a whole, this indicates that in dorsal raphe, and perhaps other neurons, the site of the critical phosphorylation may be on the G protein and possibly at the GTP binding site.

  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

    Kankainen, Matti; Ojala, Teija; Holm, Liisa

    2012-02-15

    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. 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. 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 http://ekhidna.biocenter.helsinki.fi/poxo/blannotator/.

  19. DNA mimic proteins: functions, structures, and bioinformatic analysis.

    PubMed

    Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

    2014-05-13

    DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

  20. 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).

  1. 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).

  2. 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

  3. 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.

  4. Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

    PubMed Central

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

    2014-01-01

    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. 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

  6. 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.

  7. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    PubMed

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  8. 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...

  9. 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.

  10. 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.

  11. Average protein density is a molecular-weight-dependent function.

    PubMed

    Fischer, Hannes; Polikarpov, Igor; Craievich, Aldo F

    2004-10-01

    The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.

  12. 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

  13. A Survey of Computational Intelligence Techniques in Protein Function Prediction

    PubMed Central

    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. PMID:25574395

  14. 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.

  15. The S100 proteins in epidermis: Topology and function.

    PubMed

    Leśniak, Wiesława; Graczyk-Jarzynka, Agnieszka

    2015-12-01

    S100 proteins are small calcium binding proteins encoded by genes located in the epidermal differentiation complex (EDC). Differently to other proteins encoded by EDC genes, which are indispensable for normal epidermal differentiation, the role of S100 proteins in the epidermis remains largely unknown. Particular S100 proteins differ in their distribution in epidermal layers, skin appendages, melanocytes and Langerhans cells. Taking into account that each epidermal component consists of specialized cells with well-defined functions, such differential distribution may be indicative of the function of a given S100 protein. We used this criterion together with the survey of the current experimental data pertinent to epidermis to provide a fairly comprehensive view on the possible function of individual S100 proteins in this tissue. S100 proteins are differently expressed and, despite extensive structural homology, perform diverse functions in the epidermis. Certain S100 proteins probably ensure constant epidermal renewal and support wound healing while others act in epidermal differentiation or have a protective role. As their expression is differently affected in various skin pathologies, particular S100 proteins could be valuable diagnostic markers. S100 proteins seem to be important although not yet fully recognized epidermal constituents. Better understanding of their role in the epidermis might be helpful in designing therapies to various skin diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Basic units of protein structure, folding, and function.

    PubMed

    Berezovsky, Igor N; Guarnera, Enrico; Zheng, Zejun

    2017-09-01

    Study of the hierarchy of domain structure with alternative sets of domains and analysis of discontinuous domains, consisting of remote segments of the polypeptide chain, raised a question about the minimal structural unit of the protein domain. The hypothesis on the decisive role of the polypeptide backbone in determining the elementary units of globular proteins have led to the discovery of closed loops. It is reviewed here how closed loops form the loop-n-lock structure of proteins, providing the foundation for stability and designability of protein folds/domain and underlying their co-translational folding. Simplified protein sequences are considered here with the aim to explore the basic principles that presumably dominated the folding and stability of proteins in the early stages of structural evolution. Elementary functional loops (EFLs), closed loops with one or few catalytic residues, are, in turn, units of the protein function. They are apparent descendants of the prebiotic ring-like peptides, which gave rise to the first functional folds/domains being fused in the beginning of the evolution of protein structure. It is also shown how evolutionary relations between protein functional superfamilies and folds delineated with the help of EFLs can contribute to establishing the rules for design of desired enzymatic functions. Generalized descriptors of the elementary functions are proposed to be used as basic units in the future computational design. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. 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.

  18. Werner syndrome protein: functions in the response to DNA damage and replication stress in S-phase.

    PubMed

    Cheng, Wen-Hsing; Muftuoglu, Meltem; Bohr, Vilhelm A

    2007-09-01

    Werner syndrome (WS) is an excellent model system for the study of human aging. WRN, a nuclear protein mutated in WS, plays multiple roles in DNA metabolism. Our understanding about the metabolic regulation and function of this RecQ helicase has advanced greatly during the past decade, largely due to the availability of purified WRN protein, WRN knockdown cells, and WRN knockout mice. Recent biochemical and genetic studies indicate that WRN plays significant roles in DNA replication, DNA repair, and telomere maintenance. Interestingly, many WRN functions require handling of DNA ends during S-phase, and evidence suggests that WRN plays both upstream and downstream roles in the response to DNA damage. Future research should focus on the mechanism(s) of WRN in the regulation of the various DNA metabolism pathways and development of therapeutic approaches to treat premature aging syndromes such as WS.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Linking structural features of protein complexes and biological function

    PubMed Central

    Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

    2015-01-01

    Protein–protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure–function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. PMID:26131659

  4. 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

  5. A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein.

    PubMed

    Madden, S L; Cook, D M; Rauscher, F J

    1993-07-01

    The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-containing transcription factor (WT1). WT1 binds to the consensus sequence (5'-GCGGGGGCG-3') and represses transcription when bound to this site in vivo. The mechanism of repression is not yet defined. To investigate the mechanisms of transcriptional repression and map the domains of WT1 responsible, we constructed hybrid proteins between the yeast GAL4 1-147 DNA binding domain and WT1. Fusion of a 298 amino acid glutamine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding domain created a potent transcriptional repressor. The use of N- and C-terminal truncations of this segment demonstrated that as few as 96 amino acids were required for active repression by GAL4-WT1 hybrid proteins in NIH3T3 fibroblasts. However, the truncated GAL4-WT1 fusion proteins functioned poorly as repressors in embryonic kidney-derived 293 cells, suggesting cell type-specific requirements for transcriptional repression. Site-directed mutagenesis of the WT1 repression domain revealed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression function. Single repressor binding sites placed upstream of the transcription start site conferred WT1-mediated repression to a heterologous promoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression. Significant repression of transcription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transcription initiation. We conclude that the transcriptional repression function of WT1 is contained in the N-terminal, non-DNA binding domain of the protein and that repression can be functionally transferred to a heterologous DNA binding domain.

  6. 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

  7. On the detection of functionally coherent groups of protein domains with an extension to protein annotation

    PubMed Central

    McLaughlin, William A; Chen, Ken; Hou, Tingjun; Wang, Wei

    2007-01-01

    Background Protein domains coordinate to perform multifaceted cellular functions, and domain combinations serve as the functional building blocks of the cell. The available methods to identify functional domain combinations are limited in their scope, e.g. to the identification of combinations falling within individual proteins or within specific regions in a translated genome. Further effort is needed to identify groups of domains that span across two or more proteins and are linked by a cooperative function. Such functional domain combinations can be useful for protein annotation. Results Using a new computational method, we have identified 114 groups of domains, referred to as domain assembly units (DASSEM units), in the proteome of budding yeast Saccharomyces cerevisiae. The units participate in many important cellular processes such as transcription regulation, translation initiation, and mRNA splicing. Within the units the domains were found to function in a cooperative manner; and each domain contributed to a different aspect of the unit's overall function. The member domains of DASSEM units were found to be significantly enriched among proteins contained in transcription modules, defined as genes sharing similar expression profiles and presumably similar functions. The observation further confirmed the functional coherence of DASSEM units. The functional linkages of units were found in both functionally characterized and uncharacterized proteins, which enabled the assessment of protein function based on domain composition. Conclusion A new computational method was developed to identify groups of domains that are linked by a common function in the proteome of Saccharomyces cerevisiae. These groups can either lie within individual proteins or span across different proteins. We propose that the functional linkages among the domains within the DASSEM units can be used as a non-homology based tool to annotate uncharacterized proteins. PMID:17937820

  8. Infrared Structural Biology: Detect Functionally Important Structural Motions of Proteins

    NASA Astrophysics Data System (ADS)

    Xie, Aihua

    Proteins are dynamic. Lack of dynamic structures of proteins hampers our understanding of protein functions. Infrared structural biology (IRSB) is an emerging technology. There are several advantages of IRSB for mechanistic studies of proteins: (1) its excellent dynamic range (detecting structural motions from picoseconds to >= seconds); (2) its high structural sensitivity (detect tiny but functionally important structural motions such as proton transfer and changes in hydrogen bonding interaction); (3) its ability to detect different structural motions simultaneously. Successful development of infrared structural biology demands not only new experimental techniques (from infrared technologies to chemical synthesis and cell biology), but also new data processing (how to translate infrared signals into quantitative structural information of proteins). These topics will be discussed as well as examples of how to use IRSB to study structure-function relationship of proteins. This work was supported by NSF DBI1338097 and OCAST HR10-078.

  9. Structure, Function, and Evolution of Coronavirus Spike Proteins

    PubMed Central

    Li, Fang

    2017-01-01

    The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells. PMID:27578435

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Biases in the Experimental Annotations of Protein Function and Their Effect on Our Understanding of Protein Function Space

    PubMed Central

    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. PMID:23737737

  15. 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.

  16. 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. Copyright © 2015 John Wiley & Sons, Inc.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Protein mislocalization: mechanisms, functions and clinical applications in cancer

    PubMed Central

    Wang, Xiaohong; Li, Shulin

    2014-01-01

    The changes from normal cells to cancer cells are primarily regulated by genome instability, which foster hallmark functions of cancer through multiple mechanisms including protein mislocalization. Mislocalization of these proteins, including oncoproteins, tumor suppressors, and other cancer-related proteins, can interfere with normal cellular function and cooperatively drive tumor development and metastasis. This review describes the cancer-related effects of protein subcellular mislocalization, the related mislocalization mechanisms, and the potential application of this knowledge to cancer diagnosis, prognosis, and therapy. PMID:24709009

  3. 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.

  4. Exploring Protein Function Using the Saccharomyces Genome Database.

    PubMed

    Wong, Edith D

    2017-01-01

    Elucidating the function of individual proteins will help to create a comprehensive picture of cell biology, as well as shed light on human disease mechanisms, possible treatments, and cures. Due to its compact genome, and extensive history of experimentation and annotation, the budding yeast Saccharomyces cerevisiae is an ideal model organism in which to determine protein function. This information can then be leveraged to infer functions of human homologs. Despite the large amount of research and biological data about S. cerevisiae, many proteins' functions remain unknown. Here, we explore ways to use the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org ) to predict the function of proteins and gain insight into their roles in various cellular processes.

  5. 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.

  6. 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

  7. 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

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

    PubMed

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

    2008-05-06

    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.

  9. Novel upstream and downstream sequence elements contribute to polyadenylation efficiency

    PubMed Central

    Darmon, Sarah K.; Lutz, Carol S.

    2012-01-01

    Polyadenylation is a 3′ mRNA processing event that contributes to gene expression by affecting stability, export and translation of mRNA. Human polyadenylation signals (PAS) have core and auxiliary elements that bind polyadenylation factors upstream and downstream of the cleavage site. The majority of mRNAs do not have optimal upstream and downstream core elements and therefore auxiliary elements can aid in polyadenylation efficiency. Auxiliary elements have previously been identified and studied in a small number of mRNAs. We previously used a global approach to examine auxiliary elements to identify overrepresented motifs by a bioinformatic survey. This predicted information was used to direct our in vivo validation studies, all of which were accomplished using both a tandem in vivo polyadenylation assay and using reporter protein assays measured as luciferase activity. Novel auxiliary elements were placed in a test polyadenylation signal. An in vivo polyadenylation assay was used to determine the strength of the polyadenylation signal. All but one of the novel auxiliary elements enhanced the test polyadenylation signal. Effects of these novel auxiliary elements were also measured by a luciferase assay when placed in the 3′ UTR of a firefly luciferase reporter. Two novel downstream auxiliary elements and all of the novel upstream auxiliary elements showed an increase in reporter protein levels. Many well known auxiliary polyadenylation elements have been found to occur in multiple sets. However, in our study, multiple copies of novel auxiliary elements brought reporter protein levels as well as polyadenylation choice back to wild type levels. Structural features of these novel auxiliary elements may also affect the role of auxiliary elements. A MS2 structure placed upstream of the polyadenylation signal can affect polyadenylation in both the positive and negative direction. A large change in RNA structure by using novel complementary auxiliary element also

  10. 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.

  11. Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

    PubMed Central

    Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-01-01

    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. PMID:22046139

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Nanoindentation of gold nanoparticles functionalized with proteins.

    PubMed

    Wampler, Heeyeon P; Ivanisevic, Albena

    2009-06-01

    The hardness and Young's modulus of 10 and 20 nm gold nanoparticles (Au NPs) modified with bovine serum albumin and streptavidin were measured using a nanoindenter. The Au NPs were immobilized on a semiconductor surface through organic self-assembled monolayers. Changes in mechanical properties occurred when the Au NPs were immobilized on the surface. The hardness and Young's modulus were dependent on the size of the NPs, and the proteins on the particles showed highly plastic and elastic behavior compared to flat surfaces modified with self-assembled monolayers.

  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. Aeroacoustic catastrophes: upstream cusp beaming in Lilley's equation.

    PubMed

    Stone, J T; Self, R H; Howls, C J

    2017-05-01

    The downstream propagation of high-frequency acoustic waves from a point source in a subsonic jet obeying Lilley's equation is well known to be organized around the so-called 'cone of silence', a fold catastrophe across which the amplitude may be modelled uniformly using Airy functions. Here we show that acoustic waves not only unexpectedly propagate upstream, but also are organized at constant distance from the point source around a cusp catastrophe with amplitude modelled locally by the Pearcey function. Furthermore, the cone of silence is revealed to be a cross-section of a swallowtail catastrophe. One consequence of these discoveries is that the peak acoustic field upstream is not only structurally stable but also at a similar level to the known downstream field. The fine structure of the upstream cusp is blurred out by distributions of symmetric acoustic sources, but peak upstream acoustic beaming persists when asymmetries are introduced, from either arrays of discrete point sources or perturbed continuum ring source distributions. These results may pose interesting questions for future novel jet-aircraft engine designs where asymmetric source distributions arise.

  19. 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

  20. 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.

  1. 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

  2. Exploring Protein Dynamics Space: The Dynasome as the Missing Link between Protein Structure and Function

    PubMed Central

    Hensen, Ulf; Meyer, Tim; Haas, Jürgen; Rex, René; Vriend, Gert; Grubmüller, Helmut

    2012-01-01

    Proteins are usually described and classified according to amino acid sequence, structure or function. Here, we develop a minimally biased scheme to compare and classify proteins according to their internal mobility patterns. This approach is based on the notion that proteins not only fold into recurring structural motifs but might also be carrying out only a limited set of recurring mobility motifs. The complete set of these patterns, which we tentatively call the dynasome, spans a multi-dimensional space with axes, the dynasome descriptors, characterizing different aspects of protein dynamics. The unique dynamic fingerprint of each protein is represented as a vector in the dynasome space. The difference between any two vectors, consequently, gives a reliable measure of the difference between the corresponding protein dynamics. We characterize the properties of the dynasome by comparing the dynamics fingerprints obtained from molecular dynamics simulations of 112 proteins but our approach is, in principle, not restricted to any specific source of data of protein dynamics. We conclude that: 1. the dynasome consists of a continuum of proteins, rather than well separated classes. 2. For the majority of proteins we observe strong correlations between structure and dynamics. 3. Proteins with similar function carry out similar dynamics, which suggests a new method to improve protein function annotation based on protein dynamics. PMID:22606222

  3. Functional characterization of the adrenoleukodystrophy protein (ALDP) and disease pathogenesis.

    PubMed

    Gärtner, J; Dehmel, T; Klusmann, A; Roerig, P

    2002-11-01

    X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.

  4. 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.

  5. 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.

  6. 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.

  7. A Correlation between Protein Function and Ligand Binding Profiles

    PubMed Central

    Shortridge, Matthew D.; Bokemper, Michael; Copeland, Jennifer C.; Stark, Jaime L.; Powers, Robert

    2011-01-01

    We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally-derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure or evolutionary information, and therefore, extends our ability to analyze and functionally annotate novel genes. PMID:21366353

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  13. 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…

  14. Rheological and Functional Properties of Catfish Skin Protein Hydrolysates

    USDA-ARS?s Scientific Manuscript database

    Catfish skin is an abundant and underutilized resource that can be used as a unique protein source to make fish skin hydrolysates. The objectives of this study were to: isolating soluble and insoluble proteins from hydrolyzed catfish skin and study the chemical and functional properties of the prote...

  15. 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…

  16. Challenges in the Development of Functional Assays of Membrane Proteins

    PubMed Central

    Tiefenauer, Louis; Demarche, Sophie

    2012-01-01

    Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. The functional domain grouping of microtubule associated proteins

    PubMed Central

    Deane, Charlotte M; Wakefield, James G

    2008-01-01

    Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs. PMID:19704789

  2. 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

  3. Mechanisms and Functions of Spatial Protein Quality Control.

    PubMed

    Sontag, Emily Mitchell; Samant, Rahul S; Frydman, Judith

    2017-06-20

    A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

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

    PubMed Central

    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 (≈89%) of the original associations. PMID:14566057

  13. 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.

  14. Protein domain recurrence and order can enhance prediction of protein functions.

    PubMed

    Messih, Mario Abdel; Chitale, Meghana; Bajic, Vladimir B; Kihara, Daisuke; Gao, Xin

    2012-09-15

    Burgeoning sequencing technologies have generated massive amounts of genomic and proteomic data. Annotating the functions of proteins identified in this data has become a big and crucial problem. Various computational methods have been developed to infer the protein functions based on either the sequences or domains of proteins. The existing methods, however, ignore the recurrence and the order of the protein domains in this function inference. We developed two new methods to infer protein functions based on protein domain recurrence and domain order. Our first method, DRDO, calculates the posterior probability of the Gene Ontology terms based on domain recurrence and domain order information, whereas our second method, DRDO-NB, relies on the naïve Bayes methodology using the same domain architecture information. Our large-scale benchmark comparisons show strong improvements in the accuracy of the protein function inference achieved by our new methods, demonstrating that domain recurrence and order can provide important information for inference of protein functions. The new models are provided as open source programs at http://sfb.kaust.edu.sa/Pages/Software.aspx. dkihara@cs.purdue.edu, xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics Online.

  15. Artificial membranes for membrane protein purification, functionality and structure studies.

    PubMed

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  16. 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.

  17. 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

  18. 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.

  19. Protein intake and muscle function in older adults.

    PubMed

    Deer, Rachel R; Volpi, Elena

    2015-05-01

    We provide an update on the recent advances in nutrition research regarding the role of protein intake in the development and treatment of sarcopenia of aging. Specific muscle mass, strength and function cut-points for the diagnosis of sarcopenia have been identified. There is mounting evidence, as highlighted by multiple consensus statements, that the Recommended Dietary Allowance (0.8 g/kg body weight) may be inadequate to promote optimal health in older adults. Recent research indicates that in addition to total daily protein intake the timing of protein intake is also important to best stimulate muscle protein synthesis, and maintain muscle mass and function in older adults. Recent evidence suggests that the Recommended Dietary Allowance for protein is inadequate, and that the timing and distribution of protein consumption throughout daily meals may be as important as the total quantity. Research has continued to advance our understanding of protein's effects on muscle metabolism; however, there remains a need for large, long-term, randomized clinical trials examining whether the positive effects of dietary protein on muscle metabolism seen in acute studies will translate over the long term into gains of muscle mass, function, and the overall health of older adults.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Isolation and study of the functional properties of pea proteins.

    PubMed

    Tömösközi, S; Lásztity, R; Haraszi, R; Baticz, O

    2001-10-01

    Proteins of pea seeds were isolated after defatting with hexane using alkaline (0.1 M sodium hydroxide) extraction and acid (HCl) precipitation. Concentrates were also prepared by hexane extraction and ethanolic extraction (pH = 5). Gross chemical composition amino acid content and functional properties (solubility profile, emulsifying--and foaming properties, water--and oil absorption) were studied. The results were compared with the same parameters of soy and lupin protein products. Although the majority of functional characteristics of isolates were lower in comparison to soy isolates, pea protein concentrate and isolate could be successfully used in bakery products for enrichment in protein and improvement of biological value. Their utilization as meat protein substitute in some Frankfurter type sausages is also possibly.

  5. Control of blood proteins by functional disulfide bonds

    PubMed Central

    Butera, Diego; Cook, Kristina M.; Chiu, Joyce; Wong, Jason W. H.

    2014-01-01

    Most proteins in nature are chemically modified after they are made to control how, when, and where they function. The 3 core features of proteins are posttranslationally modified: amino acid side chains can be modified, peptide bonds can be cleaved or isomerized, and disulfide bonds can be cleaved. Cleavage of peptide bonds is a major mechanism of protein control in the circulation, as exemplified by activation of the blood coagulation and complement zymogens. Cleavage of disulfide bonds is emerging as another important mechanism of protein control in the circulation. Recent advances in our understanding of control of soluble blood proteins and blood cell receptors by functional disulfide bonds is discussed as is how these bonds are being identified and studied. PMID:24523239

  6. 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.

  7. 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.

  8. Growing functional modules from a seed protein via integration of protein interaction and gene expression data.

    PubMed

    Maraziotis, Ioannis A; Dimitrakopoulou, Konstantina; Bezerianos, Anastasios

    2007-10-23

    Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI) networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules) in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Usher protein functions in hair cells and photoreceptors.

    PubMed

    Cosgrove, Dominic; Zallocchi, Marisa

    2014-01-01

    The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Automatic annotation of protein motif function with Gene Ontology terms

    PubMed Central

    Lu, Xinghua; Zhai, Chengxiang; Gopalakrishnan, Vanathi; Buchanan, Bruce G

    2004-01-01

    Background Conserved protein sequence motifs are short stretches of amino acid sequence patterns that potentially encode the function of proteins. Several sequence pattern searching algorithms and programs exist foridentifying candidate protein motifs at the whole genome level. However, amuch needed and importanttask is to determine the functions of the newly identified protein motifs. The Gene Ontology (GO) project is an endeavor to annotate the function of genes or protein sequences with terms from a dynamic, controlled vocabulary and these annotations serve well as a knowledge base. Results This paperpresents methods to mine the GO knowledge base and use the association between the GO terms assigned to a sequence and the motifs matched by the same sequence as evidence for predicting the functions of novel protein motifs automatically. The task of assigning GO terms to protein motifsis viewed as both a binary classification and information retrieval problem, where PROSITE motifs are used as samples for mode training and functional prediction. The mutual information of a motif and aGO term association isfound to be a very useful feature. We take advantageof the known motifs to train a logistic regression classifier, which allows us to combine mutual information with other frequency-based features and obtain a probability of correctassociation. The trained logistic regression model has intuitively meaningful and logically plausible parameter values, and performs very well empirically according to our evaluation criteria. Conclusions In this research, different methods for automatic annotation of protein motifs have been investigated. Empirical result demonstrated that the methods have a great potential for detecting and augmenting information about thefunctions of newly discovered candidate protein motifs. PMID:15345032

  15. 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

  16. 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

  17. 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.

  18. Average protein density is a molecular-weight-dependent function

    PubMed Central

    Fischer, Hannes; Polikarpov, Igor; Craievich, Aldo F.

    2004-01-01

    The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density. PMID:15388866

  19. 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.

  20. 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.

  1. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

    PubMed Central

    2010-01-01

    Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST) method uses all-against-all substructure comparison to determine Substructural Clusters (SCs). SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH) framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated, statistically rigorous procedure

  2. 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.

  3. Protein mechanics: from single molecules to functional biomaterials.

    PubMed

    Li, Hongbin; Cao, Yi

    2010-10-19

    Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of

  4. A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

    PubMed Central

    Araya, Carlos L.; Fowler, Douglas M.; Chen, Wentao; Muniez, Ike; Kelly, Jeffery W.; Fields, Stanley

    2012-01-01

    The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein’s structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein’s function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered. PMID:23035249

  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. 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.

  7. 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.

  8. Structure and function of heterotrimeric G proteins in plants.

    PubMed

    Fujisawa, Y; Kato, H; Iwasaki, Y

    2001-08-01

    Heterotrimeric G proteins are mediators that transmit the external signals via receptor molecules to effector molecules. The G proteins consist of three different subunits: alpha, beta, and gamma subunits. The cDNAs or genes for all the alpha, beta, and gamma subunits have been isolated from many plant species, which has contributed to great progress in the study of the structure and function of the G proteins in plants. In addition, rice plants lacking the alpha subunit were generated by the antisense method and a rice mutant, Daikoku d1, was found to have mutation in the alpha-subunit gene. Both plants show abnormal morphology such as dwarfism, dark green leaf, and small round seed. The findings revealed that the G proteins are functional molecules regulating some body plans in plants. There is evidence that the plant G proteins participate at least in signaling of gibberellin at low concentrations. In this review, we summarize the currently known information on the structure of plant heterotrimeric G proteins and discuss the possible functions of the G proteins in plants.

  9. 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.

  10. 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.

  11. 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

  12. An Atomistic Statistically Effective Energy Function for Computational Protein Design.

    PubMed

    Topham, Christopher M; Barbe, Sophie; André, Isabelle

    2016-08-09

    Shortcomings in the definition of effective free-energy surfaces of proteins are recognized to be a major contributory factor responsible for the low success rates of existing automated methods for computational protein design (CPD). The formulation of an atomistic statistically effective energy function (SEEF) suitable for a wide range of CPD applications and its derivation from structural data extracted from protein domains and protein-ligand complexes are described here. The proposed energy function comprises nonlocal atom-based and local residue-based SEEFs, which are coupled using a novel atom connectivity number factor to scale short-range, pairwise, nonbonded atomic interaction energies and a surface-area-dependent cavity energy term. This energy function was used to derive additional SEEFs describing the unfolded-state ensemble of any given residue sequence based on computed average energies for partially or fully solvent-exposed fragments in regions of irregular structure in native proteins. Relative thermal stabilities of 97 T4 bacteriophage lysozyme mutants were predicted from calculated energy differences for folded and unfolded states with an average unsigned error (AUE) of 0.84 kcal mol(-1) when compared to experiment. To demonstrate the utility of the energy function for CPD, further validation was carried out in tests of its capacity to recover cognate protein sequences and to discriminate native and near-native protein folds, loop conformers, and small-molecule ligand binding poses from non-native benchmark decoys. Experimental ligand binding free energies for a diverse set of 80 protein complexes could be predicted with an AUE of 2.4 kcal mol(-1) using an additional energy term to account for the loss in ligand configurational entropy upon binding. The atomistic SEEF is expected to improve the accuracy of residue-based coarse-grained SEEFs currently used in CPD and to extend the range of applications of extant atom-based protein statistical

  13. 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

  14. 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.

  15. 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

  16. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  17. 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

  18. Diversity, classification and function of the plant protein kinase superfamily.

    PubMed

    Lehti-Shiu, Melissa D; Shiu, Shin-Han

    2012-09-19

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.

  19. 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

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

    PubMed

    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; Backes, Wayne L

    2016-04-01

    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. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. 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

  2. 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

  3. 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.

  4. Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

    PubMed

    Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

    2017-05-01

    The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 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.

  6. ACC-FMD: ant colony clustering for functional module detection in protein-protein interaction networks.

    PubMed

    Ji, Junzhong; Liu, Hongxin; Zhang, Aidong; Liu, Zhijun; Liu, Chunnian

    2015-01-01

    Mining functional modules in Protein-Protein Interaction (PPI) networks is a very important research for revealing the structure-functionality relationships in biological processes. More recently, some swarm intelligence algorithms have been successfully applied in the field. This paper presents a new nature-inspired approach, ACC-FMD, which is based on ant colony clustering to detect functional modules. First, some proteins with the higher clustering coefficients are, respectively, selected as ant seed nodes. And then, the picking and dropping operations based on ant probabilistic models are developed and employed to assign proteins into the corresponding clusters represented by seeds. Finally, the best clustering result in each generation is used to perform the information transmission by updating the similarly function. Experimental results on some benchmarked datasets show that ACC-FMD outperforms the CFinder and MCODE algorithms and has comparative performance with the MINE, COACH, DPClus and Core algorithms in terms of the general evaluation metrics.

  7. 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.

  8. PredictProtein--an open resource for online prediction of protein structural and functional features.

    PubMed

    Yachdav, Guy; Kloppmann, Edda; Kajan, Laszlo; Hecht, Maximilian; Goldberg, Tatyana; Hamp, Tobias; Hönigschmid, Peter; Schafferhans, Andrea; Roos, Manfred; Bernhofer, Michael; Richter, Lothar; Ashkenazy, Haim; Punta, Marco; Schlessinger, Avner; Bromberg, Yana; Schneider, Reinhard; Vriend, Gerrit; Sander, Chris; Ben-Tal, Nir; Rost, Burkhard

    2014-07-01

    PredictProtein is a meta-service for sequence analysis that has been predicting structural and functional features of proteins since 1992. Queried with a protein sequence it returns: multiple sequence alignments, predicted aspects of structure (secondary structure, solvent accessibility, transmembrane helices (TMSEG) and strands, coiled-coil regions, disulfide bonds and disordered regions) and function. The service incorporates analysis methods for the identification of functional regions (ConSurf), homology-based inference of Gene Ontology terms (metastudent), comprehensive subcellular localization prediction (LocTree3), protein-protein binding sites (ISIS2), protein-polynucleotide binding sites (SomeNA) and predictions of the effect of point mutations (non-synonymous SNPs) on protein function (SNAP2). Our goal has always been to develop a system optimized to meet the demands of experimentalists not highly experienced in bioinformatics. To this end, the PredictProtein results are presented as both text and a series of intuitive, interactive and visually appealing figures. The web server and sources are available at http://ppopen.rostlab.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. 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.

  10. High Resolution Mapping of Protein Sequence–Function Relationships

    PubMed Central

    Fowler, Douglas M.; Araya, Carlos L.; Fleishman, Sarel J.; Kellogg, Elizabeth H.; Stephany, Jason J.; Baker, David; Fields, Stanley

    2010-01-01

    We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity, and high throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position possessed unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence. PMID:20711194

  11. Inferring plant microRNA functional similarity using a weighted protein-protein interaction network.

    PubMed

    Meng, Jun; Liu, Dong; Luan, Yushi

    2015-11-04

    MiRNAs play a critical role in the response of plants to abiotic and biotic stress. However, the functions of most plant miRNAs remain unknown. Inferring these functions from miRNA functional similarity would thus be useful. This study proposes a new method, called PPImiRFS, for inferring miRNA functional similarity. The functional similarity of miRNAs was inferred from the functional similarity of their target gene sets. A protein-protein interaction network with semantic similarity weights of edges generated using Gene Ontology terms was constructed to infer the functional similarity between two target genes that belong to two different miRNAs, and the score for functional similarity was calculated using the weighted shortest path for the two target genes through the whole network. The experimental results showed that the proposed method was more effective and reliable than previous methods (miRFunSim and GOSemSim) applied to Arabidopsis thaliana. Additionally, miRNAs responding to the same type of stress had higher functional similarity than miRNAs responding to different types of stress. For the first time, a protein-protein interaction network with semantic similarity weights generated using Gene Ontology terms was employed to calculate the functional similarity of plant miRNAs. A novel method based on calculating the weighted shortest path between two target genes was introduced.

  12. 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

  13. A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription.

    PubMed Central

    Bram, R J; Lue, N F; Kornberg, R D

    1986-01-01

    Binding sites for the GAL4-positive regulatory protein have been identified upstream of six galactose-inducible genes of Saccharomyces cerevisiae on the basis of (i) protection in DNAse I footprints, (ii) loss of protection when excess GAL4-binding oligonucleotide is added and (iii) homology with a 23-bp dyad-symmetric consensus sequence. Many of the binding sites have been shown to function as upstream activating sequences. The number of binding sites upstream of the various genes ranges from one to four, but a feature is conserved: in cases of multiple sites there is a pair with highest binding affinity located at dyad--dyad distances of 82--87 bp. We suggest that a pair of sites facilitates repression by the GAL80-negative regulatory protein, on the basis of (i) a correlation of a pair of sites (or only one) with full (or only partial) repression and (ii) the introduction of a second site abolishing transcription occurring with one. Images Fig. 2. Fig. 3. Fig. 4. PMID:3011415

  14. 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

  15. 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." Copyright © 2014 Elsevier B.V. All rights reserved.

  16. 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.

  17. 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.

  18. Optimizing high performance computing workflow for protein functional annotation

    PubMed Central

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

    2014-01-01

    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. PMID:25313296

  19. 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.

  20. An overview of in silico protein function prediction.

    PubMed

    Sleator, Roy D; Walsh, Paul

    2010-03-01

    As the protein databases continue to expand at an exponential rate, fed by daily uploads from multiple large scale genomic and metagenomic projects, the problem of assigning a function to each new protein has become the focus of significant research interest in recent times. Herein, we review the most recent advances in the field of automated function prediction (AFP). We begin by defining what is meant by biological "function" and the means of describing such functions using standardised machine readable ontologies. We then focus on the various function-prediction programs available, both sequence and structure based, and outline their associated strengths and weaknesses. Finally, we conclude with a brief overview of the future challenges and outstanding questions in the field, which still remain unanswered.

  1. 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.

  2. [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.

  3. Leading edge cooling by upstream injection

    NASA Technical Reports Server (NTRS)

    Piva, R.

    1971-01-01

    A leading edge cooling system by upstream along the surface was investigated. The purpose of this system is to keep the leading edge below a desired temperature without excessively increasing the radius of the tip and consequently the total pressure losses. An experimental investigation was conducted to find the optimum conditions for the cooling from the point of view of upstream jet penetration and minimum shock losses. A theoretical analysis was performed to study the flow field in the mixing region between the two counterflowing streams and the results obtained compare favorably with the experimental results.

  4. 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.

  5. Utilizing natural diversity to evolve protein function: applications towards thermostability.

    PubMed

    Cole, Megan F; Gaucher, Eric A

    2011-06-01

    Protein evolution relies on designing a library of sequences that capture meaningful functional diversity in a limited number of protein variants. Several approaches take advantage of the sequence space already explored through natural selection by incorporating sequence diversity available from modern genomes (and their ancestors) when designing these libraries. The success of these approaches is, partly, owing to the fact that modern sequence diversity has already been subjected to evolutionary selective forces and thus the diversity has already been deemed 'fit to survive'. Five of these approaches will be discussed in this review to highlight how protein engineers can use evolutionary sequence history/diversity of homologous proteins in unique ways to design protein libraries. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. 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.

  7. Protein Function Prediction Using Deep Restricted Boltzmann Machines

    PubMed Central

    Zou, Xianchun; Wang, Guijun

    2017-01-01

    Accurately annotating biological functions of proteins is one of the key tasks in the postgenome era. Many machine learning based methods have been applied to predict functional annotations of proteins, but this task is rarely solved by deep learning techniques. Deep learning techniques recently have been successfully applied to a wide range of problems, such as video, images, and nature language processing. Inspired by these successful applications, we investigate deep restricted Boltzmann machines (DRBM), a representative deep learning technique, to predict the missing functional annotations of partially annotated proteins. Experimental results on Homo sapiens, Saccharomyces cerevisiae, Mus musculus, and Drosophila show that DRBM achieves better performance than other related methods across different evaluation metrics, and it also runs faster than these comparing methods. PMID:28744460

  8. 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.

  9. Full Protein Sequence Redesign with an MMGBSA Energy Function.

    PubMed

    Gaillard, Thomas; Simonson, Thomas

    2017-10-10

    Computational protein design aims to create proteins with novel properties. A key element is the energy or scoring function used to select the sequences and conformations. We study the performance of an "MMGBSA" energy function, which combines molecular mechanics terms, a generalized Born and surface area (GBSA) solvent model, with approximations that make the model pairwise additive. Our approach is implemented in the Proteus software. The use of a physics-based energy function ensures a certain model transferability and explanatory power. As a first test, we redesign the sequence of nine proteins, one position at a time, with the rest of the protein having its native sequence and crystallographic conformation. As a second test, all positions are designed together. The contributions of individual energy terms are evaluated, and various parametrizations are compared. We find that the GB term significantly improves the results compared to simple Coulomb electrostatics but is affected by pairwise decomposition errors when all positions are designed together. The SA term, with distinct energy coefficients for nonpolar and polar atoms, makes a decisive contribution to obtain realistic protein sequences and can partially compensate for the absence of a GB term. With the best GBSA protocol, we obtain nativelike protein cores and Superfamily recognition of almost all of our sequences.

  10. Structural and functional aspects of PR-10 proteins.

    PubMed

    Fernandes, Humberto; Michalska, Karolina; Sikorski, Michal; Jaskolski, Mariusz

    2013-03-01

    Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so-called pathogenesis-related (PR) proteins. For PR proteins of class-10 (PR-10), the biological function remains unclear, despite two decades of scientific research. PR-10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR-10 proteins, both in the crystalline state by X-ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR-10 function is still limited. PR-10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel β strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR-10 subclass cause allergic reactions in humans. © 2013 The Authors Journal compilation © 2013 FEBS.

  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. Functionally specified protein signatures distinctive for each of the different blue copper proteins.

    PubMed

    Giri, Anuradha Vivekanandan; Anishetty, Sharmila; Gautam, Pennathur

    2004-09-09

    Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific

  13. 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

  14. 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

  15. Characterisation and functional properties of watermelon (Citrullus lanatus) seed proteins.

    PubMed

    Wani, Ali Abas; Sogi, Dalbir Singh; Singh, Preeti; Wani, Idrees Ahmed; Shivhare, Uma S

    2011-01-15

    People in developing countries depend largely on non-conventional protein sources to augment the availability of proteins in their diets. Watermelon seed meal is reported to contain an adequate amount of nutritional proteins that could be extracted for use as nutritional ingredients in food products. Osborne classification showed that globulin was the major protein (≥500 g kg (-1)) present in watermelon seed meal, followed by albumin and glutelin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides had low molecular weights ranging from 35 to 47 kDa. Isoelectric focusing revealed that the isoelectric point of most proteins was in the acidic range 4-6. These proteins are rich in aspartic acid, glutamic acid and serine. An increase in pH (5-9) significantly (P < 0.05) decreased the denaturation enthalpy of these proteins. Among functional properties, albumin exhibited a much higher dispersibility index (810.3-869.6 g kg(-1)) than globulin (227.8-245.4 g kg(-1)), glutelin (182.1-187.7 g kg(-1)) and prolamin (162.3-177.7 g kg(-1)). Digestibility was in the ranges 760.6-910.0 and 765.5-888.5 g kg(-1) for Mateera and Sugar Baby watermelon protein fractions respectively, while surface hydrophobicity ranged from 126.4 to 173.2 and from 125.8 to 169.3 respectively. The foaming and emulsifying properties of albumin were better than those of the other proteins studied. The good nutritional and functional properties of watermelon seed meal proteins suggest their potential use in food formulations. Copyright © 2010 Society of Chemical Industry.

  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. 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.

  18. 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.

  19. 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

  20. 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.

  1. δ-Tonoplast intrinsic protein defines unique plant vacuole functions

    PubMed Central

    Jauh, Guang-Yuh; Fischer, Andreas M.; Grimes, Howard D.; Ryan, Clarence A.; Rogers, John C.

    1998-01-01

    Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), δ-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of δ-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity. PMID:9789029

  2. Deducing protein function by forensic integrative cell biology.

    PubMed

    Earnshaw, William C

    2013-12-01

    Our ability to sequence genomes has provided us with near-complete lists of the proteins that compose cells, tissues, and organisms, but this is only the beginning of the process to discover the functions of cellular components. In the future, it's going to be crucial to develop computational analyses that can predict the biological functions of uncharacterised proteins. At the same time, we must not forget those fundamental experimental skills needed to confirm the predictions or send the analysts back to the drawing board to devise new ones.

  3. 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.

  4. A new protein-protein docking scoring function based on interface residue properties.

    PubMed

    Bernauer, J; Azé, J; Janin, J; Poupon, A

    2007-03-01

    Protein-protein complexes are known to play key roles in many cellular processes. However, they are often not accessible to experimental study because of their low stability and difficulty to produce the proteins and assemble them in native conformation. Thus, docking algorithms have been developed to provide an in silico approach of the problem. A protein-protein docking procedure traditionally consists of two successive tasks: a search algorithm generates a large number of candidate solutions, and then a scoring function is used to rank them. To address the second step, we developed a scoring function based on a Voronoï tessellation of the protein three-dimensional structure. We showed that the Voronoï representation may be used to describe in a simplified but useful manner, the geometric and physico-chemical complementarities of two molecular surfaces. We measured a set of parameters on native protein-protein complexes and on decoys, and used them as attributes in several statistical learning procedures: a logistic function, Support Vector Machines (SVM), and a genetic algorithm. For the later, we used ROGER, a genetic algorithm designed to optimize the area under the receiver operating characteristics curve. To further test the scores derived with ROGER, we ranked models generated by two different docking algorithms on targets of a blind prediction experiment, improving in almost all cases the rank of native-like solutions. http://genomics.eu.org/spip/-Bioinformatics-tools-

  5. Automated protein function prediction--the genomic challenge.

    PubMed

    Friedberg, Iddo

    2006-09-01

    Overwhelmed with genomic data, biologists are facing the first big post-genomic question--what do all genes do? First, not only is the volume of pure sequence and structure data growing, but its diversity is growing as well, leading to a disproportionate growth in the number of uncharacterized gene products. Consequently, established methods of gene and protein annotation, such as homology-based transfer, are annotating less data and in many cases are amplifying existing erroneous annotation. Second, there is a need for a functional annotation which is standardized and machine readable so that function prediction programs could be incorporated into larger workflows. This is problematic due to the subjective and contextual definition of protein function. Third, there is a need to assess the quality of function predictors. Again, the subjectivity of the term 'function' and the various aspects of biological function make this a challenging effort. This article briefly outlines the history of automated protein function prediction and surveys the latest innovations in all three topics.

  6. 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.

  7. [Structural and Functional Studies on Photoactive Retinal Proteins: Light Becomes Drugs with Proteins].

    PubMed

    Sudo, Yuki

    2016-01-01

    Retinal proteins possess vitamin A aldehyde (retinal) as a chromophore within seven transmembrane α-helices. Visible light absorption of them triggers trans-cis photoisomerization of the retinal chromophore and induces structural changes in the protein moiety, resulting in a variety of biological functions such as vision, ion transportation, and photosensing. Environmental genomics revealed that retinal proteins are widely distributed through all three biological kingdoms, eukarya, bacteria, and archaea, indicating the biological significance of their light energy conversion. In addition to their biological aspect, retinal proteins have become a focus of interest in part because of applications for optogenetics. On the basis of our results and other findings, we highlight the recent progress in structural and functional studies on retinal proteins.

  8. 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.

  9. 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