Comparative proteomics reveal diverse functions and dynamic changes of Bombyx mori silk proteins spun from different development stages.

PubMed

Dong, Zhaoming; Zhao, Ping; Wang, Chen; Zhang, Yan; Chen, Jianping; Wang, Xin; Lin, Ying; Xia, Qingyou

2013-11-01

Silkworms (Bombyx mori) produce massive amounts of silk proteins to make cocoons during the final stages of larval development. Although the major components, fibroin and sericin, have been the focus for a long time, few researchers have realized the complexity of the silk proteome. We collected seven kinds of silk fibers spun by silkworm larvae at different developmental stages: the silks spun by new hatched larvae, second instar day 0 larvae, third instar day 0 larvae, fourth instar day 0 larvae, and fourth instar molting larvae, the scaffold silk used to attach the cocoon to the substrate and the cocoon silk. Analysis by liquid chromatography-tandem mass spectrometry identified 500 proteins from the seven silks. In addition to the expected fibroins, sericins, and some known protease inhibitors, we also identified further protease inhibitors, enzymes, proteins of unknown function, and other proteins. Unsurprisingly, our quantitative results showed fibroins and sericins were the most abundant proteins in all seven silks. Except for fibroins and sericins, protease inhibitors, enzymes, and proteins of unknown function were more abundant than other proteins. We found significant change in silk protein compositions through development, being consistent with their different biological functions and complicated formation.

The influence of iron on the proteomic profile of Chromobacterium violaceum.

PubMed

Lima, Daniel C; Duarte, Fábio T; Medeiros, Viviane K S; Lima, Diogo B; Carvalho, Paulo C; Bonatto, Diego; Batistuzzo de Medeiros, Silvia R

2014-10-20

Chromobacterium violaceum is a bacterium commonly found in tropical and subtropical regions and is associated with important pharmacological and industrial attributes such as producing substances with therapeutic properties and synthesizing biodegradable polymers. Its genome was sequenced, however, approximately 40% of its genes still remain with unknown functions. Although C. violaceum is known by its versatile capacity of living in a wide range of environments, little is known on how it achieves such success. Here, we investigated the proteomic profile of C. violaceum cultivated in the absence and presence of high iron concentration, describing some proteins of unknown function that might play an important role in iron homeostasis, amongst others. Briefly, C. violaceum was cultivated in the absence and in the presence of 9 mM of iron during four hours. Total proteins were identified by LC-MS and through the PatternLab pipeline. Our proteomic analysis indicates major changes in the energetic metabolism, and alterations in the synthesis of key transport and stress proteins. In addition, it may suggest the presence of a yet unidentified operon that could be related to oxidative stress, together with a set of other proteins with unknown function. The protein-protein interaction network also pinpointed the importance of energetic metabolism proteins to the acclimatation of C. violaceum in high concentration of iron. This is the first proteomic analysis of the opportunistic pathogen C. violaceum in the presence of high iron concentration. Our data allowed us to identify a yet undescribed operon that might have a role in oxidative stress defense. Our work provides new data that will contribute to understand how this bacterium achieve its capacity of surviving in harsh conditions as well as to open a way to explore the yet little availed biotechnological characteristics of this bacterium with the further exploring of the proteins of unknown function that we showed to be up-regulated in high iron concentration.

The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mann, Greg; Koehnke, Jesko; Bent, Andrew F.

The highly conserved domain of unknown function in the cyanobactin superfamily has a novel fold. The protein does not appear to bind the most plausible substrates, leaving questions as to its role. Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological ‘toolkit’ to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized.more » The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated.« less

Proteins of Unknown Biochemical Function: A Persistent Problem and a Roadmap to Help Overcome It.

PubMed

Niehaus, Thomas D; Thamm, Antje M K; de Crécy-Lagard, Valérie; Hanson, Andrew D

2015-11-01

The number of sequenced genomes is rapidly increasing, but functional annotation of the genes in these genomes lags far behind. Even in Arabidopsis (Arabidopsis thaliana), only approximately 40% of enzyme- and transporter-encoding genes have credible functional annotations, and this number is even lower in nonmodel plants. Functional characterization of unknown genes is a challenge, but various databases (e.g. for protein localization and coexpression) can be mined to provide clues. If homologous microbial genes exist-and about one-half the genes encoding unknown enzymes and transporters in Arabidopsis have microbial homologs-cross-kingdom comparative genomics can powerfully complement plant-based data. Multiple lines of evidence can strengthen predictions and warrant experimental characterization. In some cases, relatively quick tests in genetically tractable microbes can determine whether a prediction merits biochemical validation, which is costly and demands specialized skills. © 2015 American Society of Plant Biologists. All Rights Reserved.

Bam35 tectivirus intraviral interaction map unveils new function and localization of phage ORFan proteins.

PubMed

Berjón-Otero, Mónica; Lechuga, Ana; Mehla, Jitender; Uetz, Peter; Salas, Margarita; Redrejo-Rodríguez, Modesto

2017-07-26

Tectiviridae comprises a group of tail-less, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria.In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing the knowledge of the viral proteome, we carried out a comprehensive yeast two-hybrid analysis among all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprises 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggested that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, being the latter also supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving non-structural proteins, such as the DNA binding protein P1 and the genome terminal protein (P4), which was confirmed by co-immunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle. IMPORTANCE Tail-less viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function.Comprehensive protein-protein interaction (PPI) analysis among viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins using multi-vector yeast two-hybrid screening that was further supported by co-immunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize on the biological role localization within the viral particle of some viral ORFan proteins that will be helpful for understanding the biology of Gram-positive tectivirus. Copyright © 2017 American Society for Microbiology.

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

PubMed Central

Berjón-Otero, Mónica; Lechuga, Ana; Mehla, Jitender; Uetz, Peter

2017-01-01

ABSTRACT The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle. IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria. PMID:28747494

The challenge of annotating protein sequences: The tale of eight domains of unknown function in Pfam.

PubMed

Goonesekere, Nalin C W; Shipely, Krysten; O'Connor, Kevin

2010-06-01

The Pfam database is an important tool in genome annotation, since it provides a collection of curated protein families. However, a subset of these families, known as domains of unknown function (DUFs), remains poorly characterized. We have related sequences from DUF404, DUF407, DUF482, DUF608, DUF810, DUF853, DUF976 and DUF1111 to homologs in PDB, within the midnight zone (9-20%) of sequence identity. These relationships were extended to provide functional annotation by sequence analysis and model building. Also described are examples of residue plasticity within enzyme active sites, and change of function within homologous sequences of a DUF. Copyright 2010 Elsevier Ltd. All rights reserved.

A set of GFP organelle marker lines for intracellular localization studies in Medicago truncatula

USDA-ARS?s Scientific Manuscript database

Genomics advances in the model legume Medicago truncatula have led to an increase in the number of identified genes encoding proteins with unknown biological function. Determining the intracellular location of uncharacterized proteins often aids in the elucidation of biological function. To expedite...

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.

Construction and Screening of a Lentiviral Secretome Library.

PubMed

Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

2017-06-22

Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mining high-throughput experimental data to link gene and function.

PubMed

Blaby-Haas, Crysten E; de Crécy-Lagard, Valérie

2011-04-01

Nearly 2200 genomes that encode around 6 million proteins have now been sequenced. Around 40% of these proteins are of unknown function, even when function is loosely and minimally defined as 'belonging to a superfamily'. In addition to in silico methods, the swelling stream of high-throughput experimental data can give valuable clues for linking these unknowns with precise biological roles. The goal is to develop integrative data-mining platforms that allow the scientific community at large to access and utilize this rich source of experimental knowledge. To this end, we review recent advances in generating whole-genome experimental datasets, where this data can be accessed, and how it can be used to drive prediction of gene function. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Analysis of virulence factors of Porphyromonas endodontalis based on comparative proteomics technique].

PubMed

Li, H; Ji, H; Wu, S S; Hou, B X

2016-12-09

Objective: To analyze the protein expression profile and the potential virulence factors of Porphyromonas endodontalis (Pe) via comparison with that of two strains of Porphyromonas gingivalis (Pg) with high and low virulences, respectively. Methods: Whole cell comparative proteomics of Pe ATCC35406 was examined and compared with that of high virulent strain Pg W83 andlow virulent strain Pg ATCC33277, respectively. Isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) were adopted to identify and quantitate the proteins of Pe and two strains of Pg with various virulences by using the methods of isotopically labeled peptides, mass spectrometric detection and bioinformatics analysis. The biological functions of similar proteins expressed by Pe ATCC35406 and two strains of Pg were quantified and analyzed. Results: Totally 1 210 proteins were identified while Pe compared with Pg W83. There were 130 proteins (10.74% of the total proteins) expressed similarly, including 89 known functional proteins and 41 proteins of unknown functions. Totally 1 223 proteins were identified when Pe compared with Pg ATCC33277. There were 110 proteins (8.99% of the total proteins) expressed similarly, including 72 known functional proteins and 38 proteins of unknown functions. The similarly expressed proteins in Pe and Pg strains with various virulences mainly focused on catalytic activity and binding function, including recombination activation gene (RagA), lipoprotein, chaperonin Dnak, Clp family proteins (ClpC and ClpX) and various iron-binding proteins. They were involved in metabolism and cellular processes. In addition, the type and number of similar virulence proteins between Pe and high virulence Pg were higher than those between Pe and low virulence Pg. Conclusions: Lipoprotein, oxygen resistance protein, iron binding protein were probably the potential virulence factors of Pe ATCC35406. It was speculated that pathogenicity of Pe was more similar to high virulence Pg than that to low virulence strain.

'Unknown' proteins and 'orphan' enzymes: the missing half of the engineering parts list--and how to find it.

PubMed

Hanson, Andrew D; Pribat, Anne; Waller, Jeffrey C; de Crécy-Lagard, Valérie

2009-12-14

Like other forms of engineering, metabolic engineering requires knowledge of the components (the 'parts list') of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of 'unknown' proteins and 'orphan' enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the 'missing parts list' problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

Analyses of pea necrotic yellow dwarf virus-encoded proteins.

PubMed

Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

2017-06-01

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

Genetics Home Reference: megalencephalic leukoencephalopathy with subcortical cysts

MedlinePlus

... is unable to correctly transport GlialCAM and MLC1 proteins to cell junctions. It is unknown how a lack of functional MLC1 or GlialCAM protein at cell junctions in the brain impairs brain development and ...

Detection of functionally important regions in "hypothetical proteins" of known structure.

PubMed

Nimrod, Guy; Schushan, Maya; Steinberg, David M; Ben-Tal, Nir

2008-12-10

Structural genomics initiatives provide ample structures of "hypothetical proteins" (i.e., proteins of unknown function) at an ever increasing rate. However, without function annotation, this structural goldmine is of little use to biologists who are interested in particular molecular systems. To this end, we used (an improved version of) the PatchFinder algorithm for the detection of functional regions on the protein surface, which could mediate its interactions with, e.g., substrates, ligands, and other proteins. Examination, using a data set of annotated proteins, showed that PatchFinder outperforms similar methods. We collected 757 structures of hypothetical proteins and their predicted functional regions in the N-Func database. Inspection of several of these regions demonstrated that they are useful for function prediction. For example, we suggested an interprotein interface and a putative nucleotide-binding site. A web-server implementation of PatchFinder and the N-Func database are available at http://patchfinder.tau.ac.il/.

A traveling salesman approach for predicting protein functions.

PubMed

Johnson, Olin; Liu, Jing

2006-10-12

Protein-protein interaction information can be used to predict unknown protein functions and to help study biological pathways. Here we present a new approach utilizing the classic Traveling Salesman Problem to study the protein-protein interactions and to predict protein functions in budding yeast Saccharomyces cerevisiae. We apply the global optimization tool from combinatorial optimization algorithms to cluster the yeast proteins based on the global protein interaction information. We then use this clustering information to help us predict protein functions. We use our algorithm together with the direct neighbor algorithm 1 on characterized proteins and compare the prediction accuracy of the two methods. We show our algorithm can produce better predictions than the direct neighbor algorithm, which only considers the immediate neighbors of the query protein. Our method is a promising one to be used as a general tool to predict functions of uncharacterized proteins and a successful sample of using computer science knowledge and algorithms to study biological problems.

A traveling salesman approach for predicting protein functions

PubMed Central

Johnson, Olin; Liu, Jing

2006-01-01

Background Protein-protein interaction information can be used to predict unknown protein functions and to help study biological pathways. Results Here we present a new approach utilizing the classic Traveling Salesman Problem to study the protein-protein interactions and to predict protein functions in budding yeast Saccharomyces cerevisiae. We apply the global optimization tool from combinatorial optimization algorithms to cluster the yeast proteins based on the global protein interaction information. We then use this clustering information to help us predict protein functions. We use our algorithm together with the direct neighbor algorithm [1] on characterized proteins and compare the prediction accuracy of the two methods. We show our algorithm can produce better predictions than the direct neighbor algorithm, which only considers the immediate neighbors of the query protein. Conclusion Our method is a promising one to be used as a general tool to predict functions of uncharacterized proteins and a successful sample of using computer science knowledge and algorithms to study biological problems. PMID:17147783

Predicting Structure and Function for Novel Proteins of an Extremophilic Iron Oxidizing Bacterium

NASA Astrophysics Data System (ADS)

Wheeler, K.; Zemla, A.; Banfield, J.; Thelen, M.

2007-12-01

Proteins isolated from uncultivated microbial populations represent the functional components of microbial processes and contribute directly to community fitness under natural conditions. Investigations into proteins in the environment are hindered by the lack of genome data, or where available, the high proportion of proteins of unknown function. We have identified thousands of proteins from biofilms in the extremely acidic drainage outflow of an iron mine ecosystem (1). With an extensive genomic and proteomic foundation, we have focused directly on the problem of several hundred proteins of unknown function within this well-defined model system. Here we describe the geobiological insights gained by using a high throughput computational approach for predicting structure and function of 421 novel proteins from the biofilm community. We used a homology based modeling system to compare these proteins to those of known structure (AS2TS) (2). This approach has resulted in the assignment of structures to 360 proteins (85%) and provided functional information for up to 75% of the modeled proteins. Detailed examination of the modeling results enables confident, high-throughput prediction of the roles of many of the novel proteins within the microbial community. For instance, one prediction places a protein in the phosphoenolpyruvate/pyruvate domain superfamily as a carboxylase that fills in a gap in an otherwise complete carbon cycle. Particularly important for a community in such a metal rich environment is the evolution of over 25% of the novel proteins that contain a metal cofactor; of these, one third are likely Fe containing proteins. Two of the most abundant proteins in biofilm samples are unusual c-type cytochromes. Both of these proteins catalyze iron- oxidation, a key metabolic reaction supporting the energy requirements of this community. Structural models of these cytochromes verify our experimental results on heme binding and electron transfer reactivity, and provide details for a working hypothesis of electron flow within the biofilm's major bacterium. Nearly 7% of the novel proteins contain tetratrico peptide repeat (TPR) modules, a protein-protein interaction domain that participates in signal transduction and a wide variety of other cellular functions. Like many biofilms, the various organisms in this community use unknown mechanisms to communicate, relying upon each other for survival. Especially interesting is evidence that most of these novel TPR proteins are located in the extracellular or membrane fractions, suggesting their role in intracellular communication. (1) Ram et al, 2005, Science 308:1915-20, "Community Proteomics of a Natural Microbial Biofilm" (2) Zemla et al, 2005, Nucleic Acids Res 33 (Web Server issue):W111-5, "AS2TS system for protein structure modeling and analysis" This work was funded by the DOE Genomics: GTL Program and was performed under the auspices of the DOE by the University of California, Lawrence Livermore National Laboratory under contract W-7405-Eng-48.

RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

PubMed

Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

2015-01-01

In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps.

PubMed

Chang, Yi-Chien; Hu, Zhenjun; Rachlin, John; Anton, Brian P; Kasif, Simon; Roberts, Richard J; Steffen, Martin

2016-01-04

The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼ 3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Crystal structures of MW1337R and lin2004: Representatives of a novel protein family that adopt a four-helical bundle fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kozbial, Piotr; Xu, Qingping; Chiu, Hsiu-Ju

2009-08-28

To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs,more » which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similarity to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures of MW1337R and lin2004, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG), part of the National Institute of General Medical Sciences Protein Structure Initiative.« less

Lifting the nebula: novel insights into skeletal muscle contractility.

PubMed

Ottenheijm, Coen A C; Granzier, Henk

2010-10-01

Nebulin is a giant protein and a constituent of the skeletal muscle sarcomere. The name of this protein refers to its unknown (i.e., nebulous) function. However, recent rapid advances reveal that nebulin plays important roles in the regulation of muscle contraction. When these functions of nebulin are compromised, muscle weakness ensues, as is the case in patients with nemaline myopathy.

Exploration of Uncharted Regions of the Protein Universe

PubMed Central

Jaroszewski, Lukasz; Li, Zhanwen; Krishna, S. Sri; Bakolitsa, Constantina; Wooley, John; Deacon, Ashley M.; Wilson, Ian A.; Godzik, Adam

2009-01-01

The genome projects have unearthed an enormous diversity of genes of unknown function that are still awaiting biological and biochemical characterization. These genes, as most others, can be grouped into families based on sequence similarity. The PFAM database currently contains over 2,200 such families, referred to as domains of unknown function (DUF). In a coordinated effort, the four large-scale centers of the NIH Protein Structure Initiative have determined the first three-dimensional structures for more than 250 of these DUF families. Analysis of the first 248 reveals that about two thirds of the DUF families likely represent very divergent branches of already known and well-characterized families, which allows hypotheses to be formulated about their biological function. The remainder can be formally categorized as new folds, although about one third of these show significant substructure similarity to previously characterized folds. These results infer that, despite the enormous increase in the number and the diversity of new genes being uncovered, the fold space of the proteins they encode is gradually becoming saturated. The previously unexplored sectors of the protein universe appear to be primarily shaped by extreme diversification of known protein families, which then enables organisms to evolve new functions and adapt to particular niches and habitats. Notwithstanding, these DUF families still constitute the richest source for discovery of the remaining protein folds and topologies. PMID:19787035

Efficacy of function specific 3D-motifs in enzyme classification according to their EC-numbers.

PubMed

Rahimi, Amir; Madadkar-Sobhani, Armin; Touserkani, Rouzbeh; Goliaei, Bahram

2013-11-07

Due to the increasing number of protein structures with unknown function originated from structural genomics projects, protein function prediction has become an important subject in bioinformatics. Among diverse function prediction methods, exploring known 3D-motifs, which are associated with functional elements in unknown protein structures is one of the most biologically meaningful methods. Homologous enzymes inherit such motifs in their active sites from common ancestors. However, slight differences in the properties of these motifs, results in variation in the reactions and substrates of the enzymes. In this study, we examined the possibility of discriminating highly related active site patterns according to their EC-numbers by 3D-motifs. For each EC-number, the spatial arrangement of an active site, which has minimum average distance to other active sites with the same function, was selected as a representative 3D-motif. In order to characterize the motifs, various points in active site elements were tested. The results demonstrated the possibility of predicting full EC-number of enzymes by 3D-motifs. However, the discriminating power of 3D-motifs varies among different enzyme families and depends on selecting the appropriate points and features. © 2013 Elsevier Ltd. All rights reserved.

Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajaj, R. Alexandra; Arbing, Mark A.; Shin, Annie

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin–antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate–latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novelmore » TA system involved inMycobacterium tuberculosislatency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.« less

Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: insights into structure and function.

PubMed

Ramakrishnan, Gayatri; Ochoa-Montaño, Bernardo; Raghavender, Upadhyayula S; Mudgal, Richa; Joshi, Adwait G; Chandra, Nagasuma R; Sowdhamini, Ramanathan; Blundell, Tom L; Srinivasan, Narayanaswamy

2015-01-01

The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better understanding of pathogenesis and to accelerate the process of drug target discovery. Copyright © 2014 Elsevier Ltd. All rights reserved.

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58*

PubMed Central

Wichelecki, Daniel J.; Vetting, Matthew W.; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T.; Almo, Steven C.; Gerlt, John A.

2015-01-01

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

Proteins with neomorphic moonlighting functions in disease.

PubMed

Jeffery, Constance J

2011-07-01

One gene can encode multiple protein functions because of RNA splice variants, gene fusions during evolution, promiscuous enzyme activities, and moonlighting protein functions. In addition to these types of multifunctional proteins, in which both functions are considered "normal" functions of a protein, some proteins have been described in which a mutation or conformational change imparts a second function on a protein that is not a "normal" function of the protein. We propose to call these new functions "neomorphic moonlighting functions". The most common examples of neomorphic moonlighting functions are due to conformational changes that impart novel protein-protein interactions resulting in the formation of protein aggregates in Alzheimers, Parkinsons disease, and the systemic amyloidoses. Other changes that can result in a neomorphic moonlighting function include a mutation in SMAD4 that causes the protein to bind to new promoters and thereby alter gene transcription patterns, mutations in two isocitrate dehydrogenase isoforms that impart a new catalytic activity, and mutations in dihydrolipoamide dehydrogenase that activate a hidden protease activity. These neomorphic moonlighting functions were identified because of their connection to disease. In the cases described herein, the new functions cause cancers or severe neurological impairment, although in most cases the mechanism by which the new function leads to disease is unknown. Copyright © 2011 Wiley Periodicals, Inc.

Comparative Metagenomics of Cellulose- and Poplar Hydrolysate-Degrading Microcosms from Gut Microflora of the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus) after Long-Term Enrichment

PubMed Central

Wong, Mabel T.; Wang, Weijun; Couturier, Marie; Razeq, Fakhria M.; Lombard, Vincent; Lapebie, Pascal; Edwards, Elizabeth A.; Terrapon, Nicolas; Henrissat, Bernard; Master, Emma R.

2017-01-01

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization. PMID:29326667

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

PubMed

Pan, Joshua; Meyers, Robin M; Michel, Brittany C; Mashtalir, Nazar; Sizemore, Ann E; Wells, Jonathan N; Cassel, Seth H; Vazquez, Francisca; Weir, Barbara A; Hahn, William C; Marsh, Joseph A; Tsherniak, Aviad; Kadoch, Cigall

2018-05-23

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

SVM-Prot 2016: A Web-Server for Machine Learning Prediction of Protein Functional Families from Sequence Irrespective of Similarity.

PubMed

Li, Ying Hong; Xu, Jing Yu; Tao, Lin; Li, Xiao Feng; Li, Shuang; Zeng, Xian; Chen, Shang Ying; Zhang, Peng; Qin, Chu; Zhang, Cheng; Chen, Zhe; Zhu, Feng; Chen, Yu Zong

2016-01-01

Knowledge of protein function is important for biological, medical and therapeutic studies, but many proteins are still unknown in function. There is a need for more improved functional prediction methods. Our SVM-Prot web-server employed a machine learning method for predicting protein functional families from protein sequences irrespective of similarity, which complemented those similarity-based and other methods in predicting diverse classes of proteins including the distantly-related proteins and homologous proteins of different functions. Since its publication in 2003, we made major improvements to SVM-Prot with (1) expanded coverage from 54 to 192 functional families, (2) more diverse protein descriptors protein representation, (3) improved predictive performances due to the use of more enriched training datasets and more variety of protein descriptors, (4) newly integrated BLAST analysis option for assessing proteins in the SVM-Prot predicted functional families that were similar in sequence to a query protein, and (5) newly added batch submission option for supporting the classification of multiple proteins. Moreover, 2 more machine learning approaches, K nearest neighbor and probabilistic neural networks, were added for facilitating collective assessment of protein functions by multiple methods. SVM-Prot can be accessed at http://bidd2.nus.edu.sg/cgi-bin/svmprot/svmprot.cgi.

Identification of functional candidates amongst hypothetical proteins of Treponema pallidum ssp. pallidum.

PubMed

Naqvi, Ahmad Abu Turab; Shahbaaz, Mohd; Ahmad, Faizan; Hassan, Md Imtaiyaz

2015-01-01

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.

Generation and Analysis of Expressed Sequence Tags from Olea europaea L.

PubMed Central

Ozdemir Ozgenturk, Nehir; Oruç, Fatma; Sezerman, Ugur; Kuçukural, Alper; Vural Korkut, Senay; Toksoz, Feriha; Un, Cemal

2010-01-01

Olive (Olea europaea L.) is an important source of edible oil which was originated in Near-East region. In this study, two cDNA libraries were constructed from young olive leaves and immature olive fruits for generation of ESTs to discover the novel genes and search the function of unknown genes of olive. The randomly selected 3840 colonies were sequenced for EST collection from both libraries. Readable 2228 sequences for olive leaf and 1506 sequences for olive fruit were assembled into 205 and 69 contigs, respectively, whereas 2478 were singletons. Putative functions of all 2752 differentially expressed unique sequences were designated by gene homology based on BLAST and annotated using BLAST2GO. While 1339 ESTs show no homology to the database, 2024 ESTs have homology (under 80%) with hypothetical proteins, putative proteins, expressed proteins, and unknown proteins in NCBI-GenBank. 635 EST's unique genes sequence have been identified by over 80% homology to known function in other species which were not previously described in Olea family. Only 3.1% of total EST's was shown similarity with olive database existing in NCBI. This generated EST's data and consensus sequences were submitted to NCBI as valuable source for functional genome studies of olive. PMID:21197085

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2*

PubMed Central

Yazicioglu, Mustafa N.; Monaldini, Luca; Chu, Kirk; Khazi, Fayaz R.; Murphy, Samuel L.; Huang, Heshu; Margaritis, Paris; High, Katherine A.

2013-01-01

The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins. PMID:23873930

Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

USDA-ARS?s Scientific Manuscript database

Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

The T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B.

PubMed

Lapinski, Philip E; Oliver, Jennifer A; Bodie, Jennifer N; Marti, Francesc; King, Philip D

2009-11-01

Adapter proteins play key roles in intracellular signal transduction through complex formation with catalytically active signaling molecules. In T lymphocytes, the role of several different types of adapter proteins in T-cell antigen receptor signal transduction is well established. An exception to this is the family of T-cell-specific adapter (TSAd) proteins comprising of TSAd, adapter protein of unknown function (ALX), SH2D4A, and SH2D4B. Only recently has the function of these adapters in T-cell signal transduction been explored. Here, we discuss advances in our understanding of the role of this family of adapter proteins in T cells. Their function as regulators of signal transduction in other cell types is also discussed.

Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering.

PubMed

Boari de Lima, Elisa; Meira, Wagner; Melo-Minardi, Raquel Cardoso de

2016-06-01

As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem's complexity. Hence, this work's purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting subfamilies in a family with different specificities, thus reducing the complexity of the experimental function characterization problem; and the identification of residues responsible for specificity.

Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering

PubMed Central

Boari de Lima, Elisa; Meira, Wagner; de Melo-Minardi, Raquel Cardoso

2016-01-01

As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem’s complexity. Hence, this work’s purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting subfamilies in a family with different specificities, thus reducing the complexity of the experimental function characterization problem; and the identification of residues responsible for specificity. PMID:27348631

Identification of functional candidates amongst hypothetical proteins of Mycobacterium leprae Br4923, a causative agent of leprosy.

PubMed

Naqvi, Ahmad Abu Turab; Ahmad, Faizan; Hassan, Md Imtaiyaz

2015-01-01

Mycobacterium leprae is an intracellular obligate parasite that causes leprosy in humans, and it leads to the destruction of peripheral nerves and skin deformation. Here, we report an extensive analysis of the hypothetical proteins (HPs) from M. leprae strain Br4923, assigning their functions to better understand the mechanism of pathogenesis and to search for potential therapeutic interventions. The genome of M. leprae encodes 1604 proteins, of which the functions of 632 are not known (HPs). In this paper, we predicted the probable functions of 312 HPs. First, we classified all HPs into families and subfamilies on the basis of sequence similarity, followed by domain assignment, which provides many clues for their possible function. However, the functions of 320 proteins were not predicted because of low sequence similarity with proteins of known function. Annotated HPs were categorized into enzymes, binding proteins, transporters, and proteins involved in cellular processes. We found several novel proteins whose functions were unknown for M. leprae. These proteins have a requisite association with bacterial virulence and pathogenicity. Finally, our sequence-based analysis will be helpful for further validation and the search for potential drug targets while developing effective drugs to cure leprosy.

Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

PubMed

Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

2015-12-15

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

Import routes and nuclear functions of Argonaute and other small RNA-silencing proteins.

PubMed

Schraivogel, Daniel; Meister, Gunter

2014-09-01

Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

A method for partitioning the information contained in a protein sequence between its structure and function.

PubMed

Possenti, Andrea; Vendruscolo, Michele; Camilloni, Carlo; Tiana, Guido

2018-05-23

Proteins employ the information stored in the genetic code and translated into their sequences to carry out well-defined functions in the cellular environment. The possibility to encode for such functions is controlled by the balance between the amount of information supplied by the sequence and that left after that the protein has folded into its structure. We study the amount of information necessary to specify the protein structure, providing an estimate that keeps into account the thermodynamic properties of protein folding. We thus show that the information remaining in the protein sequence after encoding for its structure (the 'information gap') is very close to what needed to encode for its function and interactions. Then, by predicting the information gap directly from the protein sequence, we show that it may be possible to use these insights from information theory to discriminate between ordered and disordered proteins, to identify unknown functions, and to optimize artificially-designed protein sequences. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Agouti signaling protein stimulates cell division in "viable yellow" (A vy/a) mouse liver

USDA-ARS?s Scientific Manuscript database

Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti Avy/- mice (1); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in Avy/a or a...

Outfielders playing in the infield: functions of aging-associated "nuclear" proteins in the mitochondria.

PubMed

Czypiorski, P; Altschmied, J; Rabanter, L L; Goy, C; Jakob, S; Haendeler, J

2014-01-01

Over the past few years it has become clear that mitochondria are not merely the powerhouses of cells. Proteome-analyses of mitochondria from different organisms and organs revealed that more than 1000 proteins are localized in and/or on mitochondria. This by far exceeds the number of proteins required for classical mitochondrial functions, e.g. the respiratory chain, the tricarboxylic acid cycle, fatty acid oxidation and apoptosis. This suggests that many of these proteins have other, as yet unknown functions. Several proteins with well-described nuclear functions, like the transcription factor FoxO3A or the Telomerase Reverse Transcriptase, have recently been shown to be localized also within the mitochondria. This mini-review will focus on the description of the functions of these two proteins in the nucleus and in the mitochondria - as two examples of many more proteins, which are yet to be uncovered. It will give insights into the role of these proteins within different organelles of the cell and will reveal that the functions of the proteins are probably not the same in the nucleus and the mitochondria. Therefore, these differences have to be considered when targeting proteins for therapeutic approaches.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

PubMed Central

Vyas, Sejal; Chesarone-Cataldo, Melissa; Todorova, Tanya; Huang, Yun-Han; Chang, Paul

2013-01-01

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology. PMID:23917125

Identification of Functional Candidates amongst Hypothetical Proteins of Treponema pallidum ssp. pallidum

PubMed Central

Naqvi, Ahmad Abu Turab; Shahbaaz, Mohd; Ahmad, Faizan; Hassan, Md. Imtaiyaz

2015-01-01

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions. PMID:25894582

Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 Regulates Xylem Development and Growth by a Conserved Mechanism That Modulates Hormone Signaling1[W][OPEN

PubMed Central

Grienenberger, Etienne; Douglas, Carl J.

2014-01-01

Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways. PMID:24567189

Overexpression of a domain of unknown function 266-containing protein results in high cellulose content, reduced recalcitrance, and enhanced plant growth in the bioenergy crop Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yongil; Yoo, Chang Geun; Guo, Hao -Bo

Domain of Unknown Function 266 (DUF266) is a plant-specific domain. DUF266-containing proteins (DUF266 proteins) have been categorized as ‘not classified glycosyltransferases (GTnc)’ due to amino acid similarity with GTs. However, little is known about the function of DUF266 proteins. As a result, Phylogenetic analysis revealed that DUF266 proteins are only present in the land plants including moss and lycophyte. We report the functional characterization of one member of DUF266 proteins in Populus, PdDUF266A. PdDUF266A was ubiquitously expressed with high abundance in the xylem. In Populus transgenic plants overexpressing PdDUF266A ( OXPdDUF266A), the glucose and cellulose contents were significantly higher, whilemore » the lignin content was lower than that in the wild type. Degree of polymerization of cellulose in OXPdDUF266A transgenic plants was also higher, whereas cellulose crystallinity index remained unchanged. Gene expression analysis indicated that cellulose biosynthesis-related genes such as CESA and SUSY were upregulated in mature leaf and xylem of OXPdDUF266A transgenic plants. Moreover, PdDUF266A overexpression resulted in an increase of biomass production. Their glucose contents and biomass phenotypes were further validated via heterologous expression of PdDUF266A in Arabidopsis. Results from saccharification treatment demonstrated that the rate of sugar release was increased by approximately 38% in the OXPdDUF266A transgenic plants.« less

Overexpression of a domain of unknown function 266-containing protein results in high cellulose content, reduced recalcitrance, and enhanced plant growth in the bioenergy crop Populus

DOE PAGES

Yang, Yongil; Yoo, Chang Geun; Guo, Hao -Bo; ...

2017-03-23

Domain of Unknown Function 266 (DUF266) is a plant-specific domain. DUF266-containing proteins (DUF266 proteins) have been categorized as ‘not classified glycosyltransferases (GTnc)’ due to amino acid similarity with GTs. However, little is known about the function of DUF266 proteins. As a result, Phylogenetic analysis revealed that DUF266 proteins are only present in the land plants including moss and lycophyte. We report the functional characterization of one member of DUF266 proteins in Populus, PdDUF266A. PdDUF266A was ubiquitously expressed with high abundance in the xylem. In Populus transgenic plants overexpressing PdDUF266A ( OXPdDUF266A), the glucose and cellulose contents were significantly higher, whilemore » the lignin content was lower than that in the wild type. Degree of polymerization of cellulose in OXPdDUF266A transgenic plants was also higher, whereas cellulose crystallinity index remained unchanged. Gene expression analysis indicated that cellulose biosynthesis-related genes such as CESA and SUSY were upregulated in mature leaf and xylem of OXPdDUF266A transgenic plants. Moreover, PdDUF266A overexpression resulted in an increase of biomass production. Their glucose contents and biomass phenotypes were further validated via heterologous expression of PdDUF266A in Arabidopsis. Results from saccharification treatment demonstrated that the rate of sugar release was increased by approximately 38% in the OXPdDUF266A transgenic plants.« less

A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

PubMed

Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

2014-01-21

Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Proteomic Analysis of the Arabidopsis Nucleolus Suggests Novel Nucleolar FunctionsD⃞

PubMed Central

Pendle, Alison F.; Clark, Gillian P.; Boon, Reinier; Lewandowska, Dominika; Lam, Yun Wah; Andersen, Jens; Mann, Matthias; Lamond, Angus I.; Brown, John W. S.; Shaw, Peter J.

2005-01-01

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance. PMID:15496452

Comparative Genome Analysis of “Candidatus Phytoplasma australiense” (Subgroup tuf-Australia I; rp-A) and “Ca. Phytoplasma asteris” Strains OY-M and AY-WB▿ †

PubMed Central

Tran-Nguyen, L. T. T.; Kube, M.; Schneider, B.; Reinhardt, R.; Gibb, K. S.

2008-01-01

The chromosome sequence of “Candidatus Phytoplasma australiense” (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of “Ca. Phytoplasma australiense” with “Ca. Phytoplasma asteris” strains OY-M and AY-WB showed that the gene order was more conserved between the closely related “Ca. Phytoplasma asteris” strains than to “Ca. Phytoplasma australiense.” Differences observed between “Ca. Phytoplasma australiense” and “Ca. Phytoplasma asteris” strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function. PMID:18359806

Ror receptor tyrosine kinases: orphans no more.

PubMed

Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

2008-11-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

Defining the human deubiquitinating enzyme interaction landscape.

PubMed

Sowa, Mathew E; Bennett, Eric J; Gygi, Steven P; Harper, J Wade

2009-07-23

Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

Defining the Human Deubiquitinating Enzyme Interaction Landscape

PubMed Central

Sowa, Mathew E.; Bennett, Eric J.; Gygi, Steven P.; Harper, J. Wade

2009-01-01

Summary Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform, called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel non-reciprocal proteomic datasets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, sub-cellular localization and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway. PMID:19615732

Genetics Home Reference: Dowling-Degos disease

MedlinePlus

... for the development of normal skin pigmentation. This disruption of melanosome transport is thought to cause the ... condition are due to impaired Notch signaling or disruption of an unknown function of the protein in ...

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

PubMed Central

German, Peter; Bai, Shanshan; Liu, Xian-De; Sun, Mianen; Zhou, Lijun; Kalra, Sarathi; Zhang, Xuesong; Minelli, Rosalba; Scott, Kenneth L.; Mills, Gordon B.; Jonasch, Eric; Ding, Zhiyong

2016-01-01

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation, and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long form variant of VHL was often detected as multiple bands by Western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetic and pharmacologic approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein, thereby promoting HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases. PMID:26973240

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

PubMed

Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

2010-10-01

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Fungal proteomics: from identification to function.

PubMed

Doyle, Sean

2011-08-01

Some fungi cause disease in humans and plants, while others have demonstrable potential for the control of insect pests. In addition, fungi are also a rich reservoir of therapeutic metabolites and industrially useful enzymes. Detailed analysis of fungal biochemistry is now enabled by multiple technologies including protein mass spectrometry, genome and transcriptome sequencing and advances in bioinformatics. Yet, the assignment of function to fungal proteins, encoded either by in silico annotated, or unannotated genes, remains problematic. The purpose of this review is to describe the strategies used by many researchers to reveal protein function in fungi, and more importantly, to consolidate the nomenclature of 'unknown function protein' as opposed to 'hypothetical protein' - once any protein has been identified by protein mass spectrometry. A combination of approaches including comparative proteomics, pathogen-induced protein expression and immunoproteomics are outlined, which, when used in combination with a variety of other techniques (e.g. functional genomics, microarray analysis, immunochemical and infection model systems), appear to yield comprehensive and definitive information on protein function in fungi. The relative advantages of proteomic, as opposed to transcriptomic-only, analyses are also described. In the future, combined high-throughput, quantitative proteomics, allied to transcriptomic sequencing, are set to reveal much about protein function in fungi. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Detection and analysis of protein ISGylation.

PubMed

Takeuchi, Tomoharu; Yokosawa, Hideyoshi

2008-01-01

ISG15 is a ubiquitin-like modifi er that is conjugated to target proteins by a sequential reaction catalyzed by E1/E2/E3 enzymes (protein ISGylation). ISG15 and protein ISGylation are upregulated by interferon stimuli. ISG15 functions as an antiviral protein against Sindbis virus and HIV-1, but the molecular mechanism remains unknown. Here we describe in detail methods for detecting and analyzing protein ISGylation. The methods consist of plasmid transfection and affi nity purifi cation of ISGylated proteins. In addition, we describe a method for detecting ISGylation of a target protein, Ubc13.

Chemical Component and Proteomic Study of the Amphibalanus (= Balanus) amphitrite Shell

PubMed Central

Zhang, Gen; He, Li-sheng; Wong, Yue-Him; Xu, Ying; Zhang, Yu; Qian, Pei-yuan

2015-01-01

As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus) amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein), we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study. PMID:26222041

An Approach to Function Annotation for Proteins of Unknown Function (PUFs) in the Transcriptome of Indian Mulberry.

PubMed

Dhanyalakshmi, K H; Naika, Mahantesha B N; Sajeevan, R S; Mathew, Oommen K; Shafi, K Mohamed; Sowdhamini, Ramanathan; N Nataraja, Karaba

2016-01-01

The modern sequencing technologies are generating large volumes of information at the transcriptome and genome level. Translation of this information into a biological meaning is far behind the race due to which a significant portion of proteins discovered remain as proteins of unknown function (PUFs). Attempts to uncover the functional significance of PUFs are limited due to lack of easy and high throughput functional annotation tools. Here, we report an approach to assign putative functions to PUFs, identified in the transcriptome of mulberry, a perennial tree commonly cultivated as host of silkworm. We utilized the mulberry PUFs generated from leaf tissues exposed to drought stress at whole plant level. A sequence and structure based computational analysis predicted the probable function of the PUFs. For rapid and easy annotation of PUFs, we developed an automated pipeline by integrating diverse bioinformatics tools, designated as PUFs Annotation Server (PUFAS), which also provides a web service API (Application Programming Interface) for a large-scale analysis up to a genome. The expression analysis of three selected PUFs annotated by the pipeline revealed abiotic stress responsiveness of the genes, and hence their potential role in stress acclimation pathways. The automated pipeline developed here could be extended to assign functions to PUFs from any organism in general. PUFAS web server is available at http://caps.ncbs.res.in/pufas/ and the web service is accessible at http://capservices.ncbs.res.in/help/pufas.

Potential toxicity of graphene to cell functions via disrupting protein-protein interactions.

PubMed

Luan, Binquan; Huynh, Tien; Zhao, Lin; Zhou, Ruhong

2015-01-27

While carbon-based nanomaterials such as graphene and carbon nanotubes (CNTs) have become popular in state-of-the-art nanotechnology, their biological safety and underlying molecular mechanism is still largely unknown. Experimental studies have been focused at the cellular level and revealed good correlations between cell's death and the application of CNTs or graphene. Using large-scale all-atom molecular dynamics simulations, we theoretically investigate the potential toxicity of graphene to a biological cell at molecular level. Simulation results show that the hydrophobic protein-protein interaction (or recognition) that is essential to biological functions can be interrupted by a graphene nanosheet. Due to the hydrophobic nature of graphene, it is energetically favorable for a graphene nanosheet to enter the hydrophobic interface of two contacting proteins, such as a dimer. The forced separation of two functional proteins can disrupt the cell's metabolism and even lead to the cell's mortality.

Automated prediction of protein function and detection of functional sites from structure.

PubMed

Pazos, Florencio; Sternberg, Michael J E

2004-10-12

Current structural genomics projects are yielding structures for proteins whose functions are unknown. Accordingly, there is a pressing requirement for computational methods for function prediction. Here we present PHUNCTIONER, an automatic method for structure-based function prediction using automatically extracted functional sites (residues associated to functions). The method relates proteins with the same function through structural alignments and extracts 3D profiles of conserved residues. Functional features to train the method are extracted from the Gene Ontology (GO) database. The method extracts these features from the entire GO hierarchy and hence is applicable across the whole range of function specificity. 3D profiles associated with 121 GO annotations were extracted. We tested the power of the method both for the prediction of function and for the extraction of functional sites. The success of function prediction by our method was compared with the standard homology-based method. In the zone of low sequence similarity (approximately 15%), our method assigns the correct GO annotation in 90% of the protein structures considered, approximately 20% higher than inheritance of function from the closest homologue.

Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm1[W

PubMed Central

Matsushima, Ryo; Maekawa, Masahiko; Kusano, Miyako; Kondo, Hideki; Fujita, Naoko; Kawagoe, Yasushi; Sakamoto, Wataru

2014-01-01

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology. PMID:24335509

Rice proteome analysis: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko; Tanaka, Naoki

2005-03-01

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

PubMed Central

Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

2012-01-01

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

Mammalian proapoptotic factor ChaC1 and its homologues function as γ-glutamyl cyclotransferases acting specifically on glutathione

PubMed Central

Kumar, Akhilesh; Tikoo, Shweta; Maity, Shuvadeep; Sengupta, Shantanu; Sengupta, Sagar; Kaur, Amandeep; Kumar Bachhawat, Anand

2012-01-01

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ-glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ-glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E>Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells. PMID:23070364

Structural and Phylogenetic Analysis of a Conserved Actinobacteria-Specific Protein (ASP1; SCO1997) from Streptomyces Coelicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, B.; Sugiman-Marangos, S; Junop, M

2009-01-01

The Actinobacteria phylum represents one of the largest and most diverse groups of bacteria, encompassing many important and well-characterized organisms including Streptomyces, Bifidobacterium, Corynebacterium and Mycobacterium. Members of this phylum are remarkably diverse in terms of life cycle, morphology, physiology and ecology. Recent comparative genomic analysis of 19 actinobacterial species determined that only 5 genes of unknown function uniquely define this large phylum [1]. The cellular functions of these actinobacteria-specific proteins (ASP) are not known.

Structure of the choline-binding domain of Spr1274 in Streptococcus pneumoniae.

PubMed

Zhang, Zhenyi; Li, Wenzhe; Frolet, Cecile; Bao, Rui; di Guilmi, Anne Marie; Vernet, Thierry; Chen, Yuxing

2009-08-01

Spr1274 is a putative choline-binding protein that is bound to the cell wall of Streptococcus pneumoniae through noncovalent interactions with the choline moieties of teichoic and lipoteichoic acids. Its function is still unknown. The crystal structure of the choline-binding domain of Spr1274 (residues 44-129) was solved at 2.38 A resolution with three molecules in the asymmetric unit. It may provide a structural basis for functional analysis of choline-binding proteins.

Kinesin molecular motors: Transport pathways, receptors, and human disease

NASA Astrophysics Data System (ADS)

Goldstein, Lawrence S. B.

2001-06-01

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

Monitoring the function of membrane transport proteins in detergent-solubilized form

PubMed Central

Quick, Matthias; Javitch, Jonathan A.

2007-01-01

Transport proteins constitute ≈10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment. PMID:17360689

Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family

PubMed Central

Sinha, Sangita; Rappu, Pekka; Lange, S. C.; Mäntsälä, Pekka; Zalkin, Howard; Smith, Janet L.

1999-01-01

The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-Å crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins. PMID:10557275

NDR proteins

PubMed Central

Jones, Alan M

2010-01-01

N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

Antibody to CCDC104 is associated with a paraneoplastic antibody to CDR2 (anti-Yo).

PubMed

Totland, Cecilie; Bredholt, Geir; Haugen, Mette; Haukanes, Bjørn Ivar; Vedeler, Christian A

2010-02-01

Patients with cancer may develop paraneoplastic neurological syndromes (PNS) in which onconeural antibodies are important diagnostic findings. As the functional role of onconeural antibodies is largely unknown, insight gained by identifying associated antibodies may help to clarify the pathogenesis of the PNS. In this study, we identified patients with Yo antibodies who also had antibodies to an uncharacterized protein called coiled-coil domain-containing protein 104 (CCDC104). We found a significant association between CCDC104 and Yo antibodies (4 of 38, 10.5%), but not other onconeural antibodies (0 of 158) (P = 0.007, Fisher's exact test). The prevalence of CCDC104 antibodies was approximately similar in patients with cancer (8 of 756, 1.1%) and in healthy blood donors (2 of 300, 0.7%). CCDC104 antibodies were not associated with PNS, as this was found in only two of the ten CCDC104-positive patients. The CCDC104 protein, whose function is unknown, is expressed in various human tissues, including the brain, and is localized mainly to the nucleus, but is also found in the cytoplasm. The association between Yo and CCDC104 antibodies may indicate functional similarities.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

PubMed

Eshghi, Azad; Becam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Wunder, Elsio A; Ko, Albert I; Coppee, Jean-Yves; Goarant, Cyrille; Picardeau, Mathieu

2014-06-01

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions.

PubMed

Raleigh, David R; Marchiando, Amanda M; Zhang, Yong; Shen, Le; Sasaki, Hiroyuki; Wang, Yingmin; Long, Manyuan; Turner, Jerrold R

2010-04-01

In vitro studies have demonstrated that occludin and tricellulin are important for tight junction barrier function, but in vivo data suggest that loss of these proteins can be overcome. The presence of a heretofore unknown, yet related, protein could explain these observations. Here, we report marvelD3, a novel tight junction protein that, like occludin and tricellulin, contains a conserved four-transmembrane MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain. Phylogenetic tree reconstruction; analysis of RNA and protein tissue distribution; immunofluorescent and electron microscopic examination of subcellular localization; characterization of intracellular trafficking, protein interactions, dynamic behavior, and siRNA knockdown effects; and description of remodeling after in vivo immune activation show that marvelD3, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Although marvelD3 is able to partially compensate for occludin or tricellulin loss, it cannot fully restore function. We conclude that marvelD3, occludin, and tricellulin define the tight junction-associated MARVEL protein family. The data further suggest that these proteins are best considered as a group with both redundant and unique contributions to epithelial function and tight junction regulation.

Protein–DNA Interactions: The Story so Far and a New Method for Prediction

DOE PAGES

Jones, Susan; Thornton, Janet M.

2003-01-01

This review describes methods for the prediction of DNA binding function, and specifically summarizes a new method using 3D structural templates. The new method features the HTH motif that is found in approximately one-third of DNAbinding protein families. A library of 3D structural templates of HTH motifs was derived from proteins in the PDB. Templates were scanned against complete protein structures and the optimal superposition of a template on a structure calculated. Significance thresholds in terms of a minimum root mean squared deviation (rmsd) of an optimal superposition, and a minimum motif accessible surface area (ASA), have been calculated. Inmore » this way, it is possible to scan the template library against proteins of unknown function to make predictions about DNA-binding functionality.« less

Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity

PubMed Central

Bhattacharyya, Sanchari; Bershtein, Shimon; Yan, Jin; Argun, Tijda; Gilson, Amy I; Trauger, Sunia A; Shakhnovich, Eugene I

2016-01-01

Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization – molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between ‘E. coli’s self’ and ‘foreign’ proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness. DOI: http://dx.doi.org/10.7554/eLife.20309.001 PMID:27938662

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase.

PubMed

Shield, Alison J; Murray, Tracy P; Board, Philip G

2006-09-08

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.

Overexpression of a Domain of Unknown Function 266-containing protein results in high cellulose content, reduced recalcitrance, and enhanced plant growth in the bioenergy crop Populus.

PubMed

Yang, Yongil; Yoo, Chang Geun; Guo, Hao-Bo; Rottmann, William; Winkeler, Kimberly A; Collins, Cassandra M; Gunter, Lee E; Jawdy, Sara S; Yang, Xiaohan; Guo, Hong; Pu, Yunqiao; Ragauskas, Arthur J; Tuskan, Gerald A; Chen, Jin-Gui

2017-01-01

Domain of Unknown Function 266 (DUF266) is a plant-specific domain. DUF266-containing proteins (DUF266 proteins) have been categorized as 'not classified glycosyltransferases (GTnc)' due to amino acid similarity with GTs. However, little is known about the function of DUF266 proteins. Phylogenetic analysis revealed that DUF266 proteins are only present in the land plants including moss and lycophyte. We report the functional characterization of one member of DUF266 proteins in Populus, PdDUF266A. PdDUF266A was ubiquitously expressed with high abundance in the xylem. In Populus transgenic plants overexpressing PdDUF266A ( OXPdDUF266A ), the glucose and cellulose contents were significantly higher, while the lignin content was lower than that in the wild type. Degree of polymerization of cellulose in OXPdDUF266A transgenic plants was also higher, whereas cellulose crystallinity index remained unchanged. Gene expression analysis indicated that cellulose biosynthesis-related genes such as CESA and SUSY were upregulated in mature leaf and xylem of OXPdDUF266A transgenic plants. Moreover, PdDUF266A overexpression resulted in an increase of biomass production. Their glucose contents and biomass phenotypes were further validated via heterologous expression of PdDUF266A in Arabidopsis. Results from saccharification treatment demonstrated that the rate of sugar release was increased by approximately 38% in the OXPdDUF266A transgenic plants. These results suggest that the overexpression of PdDUF266A can increase cellulose content, reduce recalcitrance, and enhance biomass production, and that PdDUF266A is a promising target for genetic manipulation for biofuel production.

Rice proteome database: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko

2005-09-01

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a beta-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.

Viruses are a dominant driver of protein adaptation in mammals.

PubMed

Enard, David; Cai, Le; Gwennap, Carina; Petrov, Dmitri A

2016-05-17

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.

Viruses are a dominant driver of protein adaptation in mammals

PubMed Central

Enard, David; Cai, Le; Gwennap, Carina; Petrov, Dmitri A

2016-01-01

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes. DOI: http://dx.doi.org/10.7554/eLife.12469.001 PMID:27187613

Potential functions of LEA proteins from the brine shrimp Artemia franciscana - anhydrobiosis meets bioinformatics.

PubMed

Janis, Brett; Uversky, Vladimir N; Menze, Michael A

2017-10-23

Late embryogenesis abundant (LEA) proteins are a large group of anhydrobiosis-associated intrinsically disordered proteins, which are commonly found in plants and some animals. The brine shrimp Artemia franciscana is the only known animal that expresses LEA proteins from three, and not only one, different groups in its anhydrobiotic life stage. The reason for the higher complexity in the A. franciscana LEA proteome (LEAome), compared with other anhydrobiotic animals, remains mostly unknown. To address this issue, we have employed a suite of bioinformatics tools to evaluate the disorder status of the Artemia LEAome and to analyze the roles of intrinsic disorder in functioning of brine shrimp LEA proteins. We show here that A. franciscana LEA proteins from different groups are more similar to each other than one originally expected, while functional differences among members of group three are possibly larger than commonly anticipated. Our data show that although these proteins are characterized by a large variety of forms and possible functions, as a general strategy, A. franciscana utilizes glassy matrix forming LEAs concurrently with proteins that more readily interact with binding partners. It is likely that the function(s) of both types, the matrix-forming and partner-binding LEA proteins, are regulated by changing water availability during desiccation.

A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein.

PubMed

Fetterer, R H; Miska, K B; Jenkins, M C; Barfield, R C

2004-12-01

A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

Protein classification using sequential pattern mining.

PubMed

Exarchos, Themis P; Papaloukas, Costas; Lampros, Christos; Fotiadis, Dimitrios I

2006-01-01

Protein classification in terms of fold recognition can be employed to determine the structural and functional properties of a newly discovered protein. In this work sequential pattern mining (SPM) is utilized for sequence-based fold recognition. One of the most efficient SPM algorithms, cSPADE, is employed for protein primary structure analysis. Then a classifier uses the extracted sequential patterns for classifying proteins of unknown structure in the appropriate fold category. The proposed methodology exhibited an overall accuracy of 36% in a multi-class problem of 17 candidate categories. The classification performance reaches up to 65% when the three most probable protein folds are considered.

Evaluating Functional Annotations of Enzymes Using the Gene Ontology.

PubMed

Holliday, Gemma L; Davidson, Rebecca; Akiva, Eyal; Babbitt, Patricia C

2017-01-01

The Gene Ontology (GO) (Ashburner et al., Nat Genet 25(1):25-29, 2000) is a powerful tool in the informatics arsenal of methods for evaluating annotations in a protein dataset. From identifying the nearest well annotated homologue of a protein of interest to predicting where misannotation has occurred to knowing how confident you can be in the annotations assigned to those proteins is critical. In this chapter we explore what makes an enzyme unique and how we can use GO to infer aspects of protein function based on sequence similarity. These can range from identification of misannotation or other errors in a predicted function to accurate function prediction for an enzyme of entirely unknown function. Although GO annotation applies to any gene products, we focus here a describing our approach for hierarchical classification of enzymes in the Structure-Function Linkage Database (SFLD) (Akiva et al., Nucleic Acids Res 42(Database issue):D521-530, 2014) as a guide for informed utilisation of annotation transfer based on GO terms.

Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions

DOE PAGES

Martí-Arbona, Ricardo; Mu, Fangping; Nowak-Lovato, Kristy L.; ...

2014-12-18

In this study, the clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity. software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed,more » we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018. These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.« less

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

PubMed

Yamamoto, Soh; Okamoto, Tomoya; Ogasawara, Noriko; Hashimoto, Shin; Shiraishi, Tsukasa; Sato, Toyotaka; Yamamoto, Keisuke; Tsutsumi, Hiroyuki; Takano, Kenichi; Himi, Testuo; Itoh, Hideaki; Yokota, Shin-Ichi

2017-02-12

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2. Copyright © 2016 Elsevier Inc. All rights reserved.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

PubMed

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO 2 affects plant growth and productivity. CO 2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO 2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO 2 ) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO 2 responses.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

PubMed Central

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses. PMID:28184230

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

LIBP-Pred: web server for lipid binding proteins using structural network parameters; PDB mining of human cancer biomarkers and drug targets in parasites and bacteria.

PubMed

González-Díaz, Humberto; Munteanu, Cristian R; Postelnicu, Lucian; Prado-Prado, Francisco; Gestal, Marcos; Pazos, Alejandro

2012-03-01

Lipid-Binding Proteins (LIBPs) or Fatty Acid-Binding Proteins (FABPs) play an important role in many diseases such as different types of cancer, kidney injury, atherosclerosis, diabetes, intestinal ischemia and parasitic infections. Thus, the computational methods that can predict LIBPs based on 3D structure parameters became a goal of major importance for drug-target discovery, vaccine design and biomarker selection. In addition, the Protein Data Bank (PDB) contains 3000+ protein 3D structures with unknown function. This list, as well as new experimental outcomes in proteomics research, is a very interesting source to discover relevant proteins, including LIBPs. However, to the best of our knowledge, there are no general models to predict new LIBPs based on 3D structures. We developed new Quantitative Structure-Activity Relationship (QSAR) models based on 3D electrostatic parameters of 1801 different proteins, including 801 LIBPs. We calculated these electrostatic parameters with the MARCH-INSIDE software and they correspond to the entire protein or to specific protein regions named core, inner, middle, and surface. We used these parameters as inputs to develop a simple Linear Discriminant Analysis (LDA) classifier to discriminate 3D structure of LIBPs from other proteins. We implemented this predictor in the web server named LIBP-Pred, freely available at , along with other important web servers of the Bio-AIMS portal. The users can carry out an automatic retrieval of protein structures from PDB or upload their custom protein structural models from their disk created with LOMETS server. We demonstrated the PDB mining option performing a predictive study of 2000+ proteins with unknown function. Interesting results regarding the discovery of new Cancer Biomarkers in humans or drug targets in parasites have been discussed here in this sense.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA.

PubMed

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A

2016-10-07

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA*

PubMed Central

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A.

2016-01-01

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. PMID:27563064

Structure of the Get3 targeting factor in complex with its membrane protein cargo

DOE PAGES

Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang; ...

2015-03-06

Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins (GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. In this paper, we reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans themore » Get3 homodimer. Finally, our data elucidate the mechanism of TA protein recognition and shielding by Get3 and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors.« less

Network-based function prediction and interactomics: the case for metabolic enzymes.

PubMed

Janga, S C; Díaz-Mejía, J Javier; Moreno-Hagelsieb, G

2011-01-01

As sequencing technologies increase in power, determining the functions of unknown proteins encoded by the DNA sequences so produced becomes a major challenge. Functional annotation is commonly done on the basis of amino-acid sequence similarity alone. Long after sequence similarity becomes undetectable by pair-wise comparison, profile-based identification of homologs can often succeed due to the conservation of position-specific patterns, important for a protein's three dimensional folding and function. Nevertheless, prediction of protein function from homology-driven approaches is not without problems. Homologous proteins might evolve different functions and the power of homology detection has already started to reach its maximum. Computational methods for inferring protein function, which exploit the context of a protein in cellular networks, have come to be built on top of homology-based approaches. These network-based functional inference techniques provide both a first hand hint into a proteins' functional role and offer complementary insights to traditional methods for understanding the function of uncharacterized proteins. Most recent network-based approaches aim to integrate diverse kinds of functional interactions to boost both coverage and confidence level. These techniques not only promise to solve the moonlighting aspect of proteins by annotating proteins with multiple functions, but also increase our understanding on the interplay between different functional classes in a cell. In this article we review the state of the art in network-based function prediction and describe some of the underlying difficulties and successes. Given the volume of high-throughput data that is being reported the time is ripe to employ these network-based approaches, which can be used to unravel the functions of the uncharacterized proteins accumulating in the genomic databases. © 2010 Elsevier Inc. All rights reserved.

Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar

2011-03-18

Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependentmore » functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.« less

Affinity resins as new tools for identifying target proteins of ascorbic acid.

PubMed

Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

2018-02-12

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

NDR proteins: lessons learned from Arabidopsis and animal cells prompt a testable hypothesis.

PubMed

Mudgil, Yashwanti; Jones, Alan M

2010-08-01

N-myc Down Regulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDL proteins show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins.

Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding

PubMed Central

Pinto, Antônio F. M.; Moresco, James; Yates, John R.; da Silva Vaz, Itabajara; Mulenga, Albert

2016-01-01

Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick feeding phases. These data set the foundation for in depth I. scapularis tick feeding physiology and TBD transmission studies. PMID:26751078

Switching on RNA Silencing Suppressor Activity by Restoring Argonaute Binding to a Viral Protein

PubMed Central

Szabó, Edit Z.; Manczinger, Máté; Göblös, Anikó; Kemény, Lajos

2012-01-01

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor. PMID:22623784

Pan-Cancer Analysis of Mutation Hotspots in Protein Domains.

PubMed

Miller, Martin L; Reznik, Ed; Gauthier, Nicholas P; Aksoy, Bülent Arman; Korkut, Anil; Gao, Jianjiong; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

2015-09-23

In cancer genomics, recurrence of mutations in independent tumor samples is a strong indicator of functional impact. However, rare functional mutations can escape detection by recurrence analysis owing to lack of statistical power. We enhance statistical power by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. Domain mutation analysis also sharpens the functional interpretation of the impact of mutations, as domains more succinctly embody function than entire genes. By mapping mutations in 22 different tumor types to equivalent positions in multiple sequence alignments of domains, we confirm well-known functional mutation hotspots, identify uncharacterized rare variants in one gene that are equivalent to well-characterized mutations in another gene, detect previously unknown mutation hotspots, and provide hypotheses about molecular mechanisms and downstream effects of domain mutations. With the rapid expansion of cancer genomics projects, protein domain hotspot analysis will likely provide many more leads linking mutations in proteins to the cancer phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

Mutant phenotypes for thousands of bacterial genes of unknown function

DOE PAGES

Price, Morgan N.; Wetmore, Kelly M.; Waters, R. Jordan; ...

2018-05-16

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because theymore » are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Lastly, our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.« less

Mutant phenotypes for thousands of bacterial genes of unknown function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Wetmore, Kelly M.; Waters, R. Jordan

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because theymore » are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Lastly, our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.« less

3D RNA and functional interactions from evolutionary couplings

PubMed Central

Weinreb, Caleb; Riesselman, Adam; Ingraham, John B.; Gross, Torsten; Sander, Chris; Marks, Debora S.

2016-01-01

Summary Non-coding RNAs are ubiquitous, but the discovery of new RNA gene sequences far outpaces research on their structure and functional interactions. We mine the evolutionary sequence record to derive precise information about function and structure of RNAs and RNA-protein complexes. As in protein structure prediction, we use maximum entropy global probability models of sequence co-variation to infer evolutionarily constrained nucleotide-nucleotide interactions within RNA molecules, and nucleotide-amino acid interactions in RNA-protein complexes. The predicted contacts allow all-atom blinded 3D structure prediction at good accuracy for several known RNA structures and RNA-protein complexes. For unknown structures, we predict contacts in 160 non-coding RNA families. Beyond 3D structure prediction, evolutionary couplings help identify important functional interactions, e.g., at switch points in riboswitches and at a complex nucleation site in HIV. Aided by accelerating sequence accumulation, evolutionary coupling analysis can accelerate the discovery of functional interactions and 3D structures involving RNA. PMID:27087444

Selection on Network Dynamics Drives Differential Rates of Protein Domain Evolution

PubMed Central

Mannakee, Brian K.; Gutenkunst, Ryan N.

2016-01-01

The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein’s rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate within networks in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces. PMID:27380265

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

PubMed Central

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

2014-01-01

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

Structure of a Trypanosoma Brucei Alpha/Beta--Hydrolase Fold Protein With Unknown Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merritt, E.A.; Holmes, M.; Buckner, F.S.

2009-05-26

The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 {angstrom} using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the {alpha}/{beta}-hydrolase fold family. Structural superposition onto representative {alpha}/{beta}-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similaritymore » at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands {beta}6 and {beta}7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.« less

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

PubMed

Harris, Kimberly A; Zhou, Zhiyuan; Peters, Michelle L; Wilkins, Sarah G; Breaker, Ronald R

2018-06-18

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans , OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA ( ∆ole ) or OapA ( ∆oapA ) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans .

LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system

PubMed Central

Bannasch, Detlev; Mehrle, Alexander; Glatting, Karl-Heinz; Pepperkok, Rainer; Poustka, Annemarie; Wiemann, Stefan

2004-01-01

We have implemented LIFEdb (http://www.dkfz.de/LIFEdb) to link information regarding novel human full-length cDNAs generated and sequenced by the German cDNA Consortium with functional information on the encoded proteins produced in functional genomics and proteomics approaches. The database also serves as a sample-tracking system to manage the process from cDNA to experimental read-out and data interpretation. A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions. PMID:14681468

Functional role of AMP-activated protein kinase in the heart during exercise.

PubMed

Musi, Nicolas; Hirshman, Michael F; Arad, Michael; Xing, Yanqiu; Fujii, Nobuharu; Pomerleau, Jason; Ahmad, Ferhaan; Berul, Charles I; Seidman, Jon G; Tian, Rong; Goodyear, Laurie J

2005-04-11

AMP-activated protein kinase (AMPK) plays a critical role in maintaining energy homeostasis and cardiac function during ischemia in the heart. However, the functional role of AMPK in the heart during exercise is unknown. We examined whether acute exercise increases AMPK activity in mouse hearts and determined the significance of these increases by studying transgenic (TG) mice expressing a cardiac-specific dominant-negative (inactivating) AMPKalpha2 subunit. Exercise increased cardiac AMPKalpha2 activity in the wild type mice but not in TG. We found that inactivation of AMPK did not result in abnormal ATP and glycogen consumption during exercise, cardiac function assessed by heart rhythm telemetry and stress echocardiography, or in maximal exercise capacity.

Stomatin-Like Protein 2 Binds Cardiolipin and Regulates Mitochondrial Biogenesis and Function▿

PubMed Central

Christie, Darah A.; Lemke, Caitlin D.; Elias, Isaac M.; Chau, Luan A.; Kirchhof, Mark G.; Li, Bo; Ball, Eric H.; Dunn, Stanley D.; Hatch, Grant M.; Madrenas, Joaquín

2011-01-01

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function. PMID:21746876

Adaptor proteins GIR1 and GIR2. I. Interaction with the repressor GLABRA2 and regulation of root hair development.

PubMed

Wu, Renhong; Citovsky, Vitaly

2017-07-01

Plants use specialized root outgrowths, termed root hairs, to enhance acquisition of nutrients and water, help secure anchorage, and facilitate interactions with soil microbiome. One of the major regulators of this process is GLABRA2 (GL2), a transcriptional repressor of root hair differentiation. However, regulation of the GL2-function is relatively well characterized, it remains completely unknown whether GL2 itself functions in complex with other transcriptional regulators. We identified GIR1 and GIR2, a plant-specific two-member family of closely related proteins that interact with GL2. Loss-of-function mutants of GIR1 and GIR2 enhanced development of root hair whereas gain-of-function mutants repressed it. Thus, GIR1 and GIR2 might function as adaptor proteins that associate with GL2 and participate in control of root hair formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of giant Mimivirus protein functions using RNA interference

PubMed Central

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

2015-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larkin, Robert M.; Stefano, Giovanni; Ruckle, Michael E.

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria andmore » chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. Finally, we conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.« less

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

Computational Analysis of Uncharacterized Proteins of Environmental Bacterial Genome

NASA Astrophysics Data System (ADS)

Coxe, K. J.; Kumar, M.

2017-12-01

Betaproteobacteria strain CB is a gram-negative bacterium in the phylum Proteobacteria and are found naturally in soil and water. In this complex environment, bacteria play a key role in efficiently eliminating the organic material and other pollutants from wastewater. To investigate the process of pollutant removal from wastewater using bacteria, it is important to characterize the proteins encoded by the bacterial genome. Our study combines a number of bioinformatics tools to predict the function of unassigned proteins in the bacterial genome. The genome of Betaproteobacteria strain CB contains 2,112 proteins in which function of 508 proteins are unknown, termed as uncharacterized proteins (UPs). The localization of the UPs with in the cell was determined and the structure of 38 UPs was accurately predicted. These UPs were predicted to belong to various classes of proteins such as enzymes, transporters, binding proteins, signal peptides, transmembrane proteins and other proteins. The outcome of this work will help better understand wastewater treatment mechanism.

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.

Comparative proteomic analysis of alfalfa revealed new salt and drought stress-related factors involved in seed germination.

PubMed

Ma, Qiaoli; Kang, Junmei; Long, Ruicai; Zhang, Tiejun; Xiong, Junbo; Zhang, Kun; Wang, Tenghua; Yang, Qingchuan; Sun, Yan

2017-07-01

Salinity and drought are two major environmental factors that limit the growth and yield of many forage crops in semi-arid and arid regions. Alfalfa (Medicago sativa L.) is one of the most important forage crops in many countries. We aim to investigate the molecular mechanisms of alfalfa in response to salt and drought stresses in this study. Physiological and proteomic analyses were applied to examine the Zhongmu NO.3 alfalfa seed germination stage with 200 mM NaCl and 180 g·L -1 polyethylene glycol (PEG) treatments. The germination ability of the seed and the accumulation of osmotic solutes were quite different between the NaCl and PEG treatments. More than 800 protein spots were detected by proteomics technology on two-dimensional electrophoresis (2-DE) gels. The abundance of twenty-eight proteins were decreased or increased after salt and drought stress. Seventeen of these proteins were identified and classified into six functional categories through mass spectrometry (MS). The six groups involved in salt- and PEG-mediated stress included defense response, energy metabolism, protein synthesis and degradation, oxidative stress, carbohydrate metabolism-associated proteins, and unknown proteins. We discovered that some proteins related to carbohydrate metabolism and energy production increased in abundance under salt- and PEG-mediated drought stress. This demonstrates a common mechanism of energy consumption during abiotic stresses. Further study of these proteins with unknown function will provide insights into the molecular mechanisms of abiotic stress and the discovery of new candidate markers.

TaFROG encodes a Pooideae orphan protein that interacts with SnRK1 and enhances resistance to the mycotoxigenic fungus fusarium graminearum.

USDA-ARS?s Scientific Manuscript database

All genomes encode taxonomically restricted ‘orphan’ genes, most of which are of unknown function. We report the functional characterization of the orphan gene TaFROG as a component of the wheat resistance to the globally important Fusarium head blight (FHB) disease. TaFROG is taxonomically restrict...

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

PubMed

Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

2004-10-01

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

The Anaerobe-Specific Orange Protein Complex of Desulfovibrio vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ54 and a Cognate Transcriptional Regulator▿†

PubMed Central

Fiévet, Anouchka; My, Laetitia; Cascales, Eric; Ansaldi, Mireille; Pauleta, Sofia R.; Moura, Isabel; Dermoun, Zorah; Bernard, Christophe S.; Dolla, Alain; Aubert, Corinne

2011-01-01

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the σ54-RNA polymerase. We further demonstrate that the σ54-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with σ54-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the σ70-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed. PMID:21531797

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

PubMed Central

Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Lucia, Maria Dell’Anna; Dell, Giulia 'Omo; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Giulia, Maria Rizzo; Gurtner, Aymone; Piaggio, Giulia

2017-01-01

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation. PMID:27793050

The Widespread Prevalence and Functional Significance of Silk-Like Structural Proteins in Metazoan Biological Materials

PubMed Central

McDougall, Carmel; Woodcroft, Ben J.

2016-01-01

In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of biological materials with diverse morphologies. The SilkSlider predictor software developed here is available at https://github.com/wwood/SilkSlider. PMID:27415783

Hunting for low abundant redox proteins in plant plasma membranes.

PubMed

Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

2009-04-13

Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis.

PubMed

Xu, Shou-Ling; Chalkley, Robert J; Maynard, Jason C; Wang, Wenfei; Ni, Weimin; Jiang, Xiaoyue; Shin, Kihye; Cheng, Ling; Savage, Dasha; Hühmer, Andreas F R; Burlingame, Alma L; Wang, Zhi-Yong

2017-02-21

Genetic studies have shown essential functions of O-linked N -acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

PubMed

Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

2018-03-28

G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

PubMed

Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

2016-12-19

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

FLOURY ENDOSPERM7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral endosperm development in rice

PubMed Central

Zhang, Long; Ren, Yulong; Lu, Bingyue; Yang, Chunyan; Feng, Zhiming; Liu, Zhou; Chen, Jun; Ma, Weiwei; Wang, Ying; Yu, Xiaowen; Wang, Yunlong; Zhang, Wenwei; Wang, Yihua; Liu, Shijia; Wu, Fuqing; Zhang, Xin; Guo, Xiuping; Bao, Yiqun; Jiang, Ling; Wan, Jianmin

2016-01-01

In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice. PMID:26608643

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bandyopadhyay, Anindya; Kopperud, Kristin; Anderson, Gavin

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine themore » subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.« less

Chemoproteomic profiling and discovery of protein electrophiles in human cells

NASA Astrophysics Data System (ADS)

Matthews, Megan L.; He, Lin; Horning, Benjamin D.; Olson, Erika J.; Correia, Bruno E.; Yates, John R.; Dawson, Philip E.; Cravatt, Benjamin F.

2017-03-01

Activity-based protein profiling (ABPP) serves as a chemical proteomic platform to discover and characterize functional amino acids in proteins on the basis of their enhanced reactivity towards small-molecule probes. This approach, to date, has mainly targeted nucleophilic functional groups, such as the side chains of serine and cysteine, using electrophilic probes. Here we show that 'reverse-polarity' (RP)-ABPP using clickable, nucleophilic hydrazine probes can capture and identify protein-bound electrophiles in cells. Using this approach, we demonstrate that the pyruvoyl cofactor of S-adenosyl-L-methionine decarboxylase (AMD1) is dynamically controlled by intracellular methionine concentrations. We also identify a heretofore unknown modification—an N-terminally bound glyoxylyl group—in the poorly characterized protein secernin-3. RP-ABPP thus provides a versatile method to monitor the metabolic regulation of electrophilic cofactors and discover novel types of electrophilic modifications on proteins in human cells.

Selective memory generalization by spatial patterning of protein synthesis

PubMed Central

O’Donnell, Cian; Sejnowski, Terrence J.

2014-01-01

Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

Selective memory generalization by spatial patterning of protein synthesis.

PubMed

O'Donnell, Cian; Sejnowski, Terrence J

2014-04-16

Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings, we proposed a two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

A cilevirus infects ornamental hibiscus in Hawaii

PubMed Central

Melzer, Michael J.; Simbajon, Nelson; Carillo, James; Borth, Wayne B.; Freitas-Astúa, Juliana; Kitajima, Elliot W.; Neupane, Kabi R.; Hu, John S.

2013-01-01

The complete nucleotide sequence of a virus infecting ornamental hibiscus (Hibiscus sp.) in Hawaii with symptoms of green ringspots on senescing leaves was determined from double-stranded RNA isolated from symptomatic tissue. Excluding polyadenylated regions at the 3′ termini, the bipartite RNA genome was 8748 and 5019 nt in length for RNA1 and RNA2, respectively. The genome organization was typical of a cilevirus: RNA1 encoded a large replication-associated protein with methyltransferase, protease, helicase and RNA-dependent RNA polymerase domains as well as a 29-kDa protein of unknown function. RNA2 possessed five open reading frames that potentially encoded proteins with molecular masses of 15, 7, 62, 32, and 24 kDa. The 32-kDa protein is homologous to 3A movement proteins of RNA viruses; the other proteins are of unknown function. A proteome comparison revealed that this virus was 92% identical to citrus leprosis virus cytoplasmic type 2 (CiLV-C2), a recently characterized cilevirus infecting citrus with leprosis-like symptoms in Colombia. The high sequence similarity suggests that the virus described in this study could be a strain of CiLV-C2, but since the new genus Cilevirus does not have species demarcation criteria established at present, the classification of this virus infecting hibiscus is open to interpretation. This study represents the first documented case of a cilevirus established in the United States and provides insight into the diversity within the genus Cilevirus. PMID:23732930

iDBPs: a web server for the identification of DNA binding proteins.

PubMed

Nimrod, Guy; Schushan, Maya; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2010-03-01

The iDBPs server uses the three-dimensional (3D) structure of a query protein to predict whether it binds DNA. First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence. We have trained and tested the classifier on various datasets and shown that it outperformed related methods. On a dataset that reflects the fraction of DNA binding proteins (DBPs) in a proteome, the area under the ROC curve was 0.90. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies. http://idbps.tau.ac.il/

Proteomics of the Lysosome

PubMed Central

Lübke, Torben; Lobel, Peter; Sleat, David

2009-01-01

Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies. PMID:18977398

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

Exploring Mouse Protein Function via Multiple Approaches.

PubMed

Huang, Guohua; Chu, Chen; Huang, Tao; Kong, Xiangyin; Zhang, Yunhua; Zhang, Ning; Cai, Yu-Dong

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 reality.

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 reality. PMID:27846315

Multifarious Functions of the Fragile X Mental Retardation Protein.

PubMed

Davis, Jenna K; Broadie, Kendal

2017-10-01

Fragile X syndrome (FXS), a heritable intellectual and autism spectrum disorder (ASD), results from the loss of Fragile X mental retardation protein (FMRP). This neurodevelopmental disease state exhibits neural circuit hyperconnectivity and hyperexcitability. Canonically, FMRP functions as an mRNA-binding translation suppressor, but recent findings have enormously expanded its proposed roles. Although connections between burgeoning FMRP functions remain unknown, recent advances have extended understanding of its involvement in RNA, channel, and protein binding that modulate calcium signaling, activity-dependent critical period development, and the excitation-inhibition (E/I) neural circuitry balance. In this review, we contextualize 3 years of FXS model research. Future directions extrapolated from recent advances focus on discovering links between FMRP roles to determine whether FMRP has a multitude of unrelated functions or whether combinatorial mechanisms can explain its multifaceted existence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Troyer Syndrome Protein Spartin Is Mono-Ubiquitinated and Functions in EGF Receptor Trafficking

PubMed Central

Jupille, Henri; Fatheddin, Parvin; Puertollano, Rosa

2007-01-01

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia caused by mutation in the spartin (SPG20) gene, which encodes a widely expressed protein of unknown function. This mutation results in premature protein truncation and thus might signify a loss-of-function disease mechanism. In this study, we have found that spartin is mono-ubiquitinated and functions in degradation of the epidermal growth factor receptor (EGFR). Upon EGF stimulation, spartin translocates from the cytoplasm to the plasma membrane and colocalizes with internalized EGF-Alexa. Knockdown of spartin by small interfering RNA decreases the rate of EGFR degradation and also affects EGFR internalization, recycling, or both. Furthermore, overexpression of spartin results in a prominent decrease in EGFR degradation. Taken together, our data suggest that spartin is involved in the intracellular trafficking of EGFR and that impaired endocytosis may underlie the pathogenesis of Troyer syndrome. PMID:17332501

Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis

PubMed Central

Wang, Zihao; Udeshi, Namrata D.; Slawson, Chad; Compton, Philip D.; Sakabe, Kaoru; Cheung, Win D.; Shabanowitz, Jeffrey; Hunt, Donald F.; Hart, Gerald W.

2010-01-01

Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division. PMID:20068230

Mining high-throughput experimental data to link gene and function

PubMed Central

Blaby-Haas, Crysten E.; de Crécy-Lagard, Valérie

2011-01-01

Nearly 2200 genomes encoding some 6 million proteins have now been sequenced. Around 40% of these proteins are of unknown function even when function is loosely and minimally defined as “belonging to a superfamily”. In addition to in silico methods, the swelling stream of high-throughput experimental data can give valuable clues for linking these “unknowns” with precise biological roles. The goal is to develop integrative data-mining platforms that allow the scientific community at large to access and utilize this rich source of experimental knowledge. To this end, we review recent advances in generating whole-genome experimental datasets, where this data can be accessed, and how it can be used to drive prediction of gene function. PMID:21310501

Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function

USDA-ARS?s Scientific Manuscript database

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after coexpression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity is unknown....

Predicting nucleic acid binding interfaces from structural models of proteins.

PubMed

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Predictive hypotheses are ineffectual in resolving complex biochemical systems.

PubMed

Fry, Michael

2018-03-20

Scientific hypotheses may either predict particular unknown facts or accommodate previously-known data. Although affirmed predictions are intuitively more rewarding than accommodations of established facts, opinions divide whether predictive hypotheses are also epistemically superior to accommodation hypotheses. This paper examines the contribution of predictive hypotheses to discoveries of several bio-molecular systems. Having all the necessary elements of the system known beforehand, an abstract predictive hypothesis of semiconservative mode of DNA replication was successfully affirmed. However, in defining the genetic code whose biochemical basis was unclear, hypotheses were only partially effective and supplementary experimentation was required for its conclusive definition. Markedly, hypotheses were entirely inept in predicting workings of complex systems that included unknown elements. Thus, hypotheses did not predict the existence and function of mRNA, the multiple unidentified components of the protein biosynthesis machinery, or the manifold unknown constituents of the ubiquitin-proteasome system of protein breakdown. Consequently, because of their inability to envision unknown entities, predictive hypotheses did not contribute to the elucidation of cation theories remained the sole instrument to explain complex bio-molecular systems, the philosophical question of alleged advantage of predictive over accommodative hypotheses became inconsequential.

Biological Function of Ribosomal Protein L10 on Cell Behavior in Human Epithelial Ovarian Cancer

PubMed Central

Shi, Jimin; Zhang, Lingyun; Zhou, Daibing; Zhang, Jinguo; Lin, Qunbo; Guan, Wencai; Zhang, Jihong; Ren, Weimin; Xu, Guoxiong

2018-01-01

Ribosomal protein L10 (RPL10) is one of large ribosomal proteins and plays a role in Wilms' tumor and premature ovarian failure. However, the function of RPL10 in human epithelial ovarian cancer (EOC) remains unknown. The purpose of this study was to examine the expression level and function of RPL10 in EOC. RPL10 protein expression was detected by immunohistochemistry and Western blot. The association RPL10 expression with clinical features was analyzed. Loss-of-function and gain-of-function approaches were applied in cellular assays, including cell viability, migration, invasion, and apoptosis. Our study demonstrated for the first time that RPL10 was upregulated in human EOC compared with normal ovarian tissues. Knockdown of RPL10 inhibited cell viability, migration, and invasion, and increased cell apoptosis. On the contrary, upregulation of RPL10 increased cell viability, migration, invasion, and decreased cell apoptosis. Furthermore, miR-143-3p regulated RPL10 expression. Our data indicate that RPL10 is a potential tissue biomarker of patients with EOC and may be a therapeutic target of ovarian cancer. PMID:29556332

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families*

PubMed Central

Dewhurst, Henry M.; Choudhury, Shilpa; Torres, Matthew P.

2015-01-01

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. PMID:26070665

TESS: a geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites.

PubMed Central

Wallace, A. C.; Borkakoti, N.; Thornton, J. M.

1997-01-01

It is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X-ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O-His-O "catalytic triad" and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions. PMID:9385633

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Computing Prediction and Functional Analysis of Prokaryotic Propionylation.

PubMed

Wang, Li-Na; Shi, Shao-Ping; Wen, Ping-Ping; Zhou, Zhi-You; Qiu, Jian-Ding

2017-11-27

Identification and systematic analysis of candidates for protein propionylation are crucial steps for understanding its molecular mechanisms and biological functions. Although several proteome-scale methods have been performed to delineate potential propionylated proteins, the majority of lysine-propionylated substrates and their role in pathological physiology still remain largely unknown. By gathering various databases and literatures, experimental prokaryotic propionylation data were collated to be trained in a support vector machine with various features via a three-step feature selection method. A novel online tool for seeking potential lysine-propionylated sites (PropSeek) ( http://bioinfo.ncu.edu.cn/PropSeek.aspx ) was built. Independent test results of leave-one-out and n-fold cross-validation were similar to each other, showing that PropSeek is a stable and robust predictor with satisfying performance. Meanwhile, analyses of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathways, and protein-protein interactions implied a potential role of prokaryotic propionylation in protein synthesis and metabolism.

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus

PubMed Central

Bukiya, Anna N.; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-01-01

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. PMID:28213520

Identification of novel transcriptional regulators of Zat12 using comprehensive yeast one-hybrid screens.

PubMed

Ben Daniel, Bat-Hen; Cattan, Esther; Wachtel, Chaim; Avrahami, Dorit; Glick, Yair; Malichy, Asaf; Gerber, Doron; Miller, Gad

2016-08-01

To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses. © 2016 Scandinavian Plant Physiology Society.

The role of saliva in tick feeding.

PubMed

Francischetti, Ivo M B; Sa-Nunes, Anderson; Mans, Ben J; Santos, Isabel M; Ribeiro, Jose M C

2009-01-01

When attempting to feed on their hosts, ticks face the problem of host hemostasis (the vertebrate mechanisms that prevent blood loss), inflammation (that can produce itching or pain and thus initiate defensive behavior on their hosts) and adaptive immunity (by way of both cellular and humoral responses). Against these barriers, ticks evolved a complex and sophisticated pharmacological armamentarium, consisting of bioactive lipids and proteins, to assist blood feeding. Recent progress in transcriptome research has uncovered that hard ticks have hundreds of different proteins expressed in their salivary glands, the majority of which have no known function, and include many novel protein families (e.g., their primary structure is unique to ticks). This review will address the vertebrate mechanisms of these barriers as a guide to identify the possible targets of these large numbers of known salivary proteins with unknown function. We additionally provide a supplemental Table that catalogues over 3,500 putative salivary proteins from various tick species, which might assist the scientific community in the process of functional identification of these unique proteins. This supplemental file is accessble fromhttp://exon.niaid.nih.gov/transcriptome/tick_review/Sup-Table-1.xls.gz.

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

PubMed Central

Uckun, Fatih M.; Ma, Hong; Zhang, Jian; Ozer, Zahide; Dovat, Sinisa; Mao, Cheney; Ishkhanian, Rita; Goodman, Patricia; Qazi, Sanjive

2012-01-01

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function. PMID:23071339

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment

DOE PAGES

Larkin, Robert M.; Stefano, Giovanni; Ruckle, Michael E.; ...

2016-02-09

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria andmore » chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. Finally, we conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.« less

Microarray and differential display identify genes involved in jasmonate-dependent anther development.

PubMed

Mandaokar, Ajin; Kumar, V Dinesh; Amway, Matt; Browse, John

2003-07-01

Jasmonate (JA) is a signaling compound essential for anther development and pollen fertility in Arabidopsis. Mutations that block the pathway of JA synthesis result into male sterility. To understand the processes of anther and pollen maturation, we used microarray and differential display approaches to compare gene expression pattern in anthers of wild-type Arabidopsis and the male-sterile mutant, opr3. Microarray experiment revealed 25 genes that were up-regulated more than 1.8-fold in wild-type anthers as compared to mutant anthers. Experiments based on differential display identified 13 additional genes up-regulated in wild-type anthers compared to opr3 for a total of 38 differentially expressed genes. Searches of the Arabidopsis and non-redundant databases disclosed known or likely functions for 28 of the 38 genes identified, while 10 genes encode proteins of unknown function. Northern blot analysis of eight representative clones as probes confirmed low expression in opr3 anthers compared with wild-type anthers. JA responsiveness of these same genes was also investigated by northern blot analysis of anther RNA isolated from wild-type and opr3 plants, In these experiments, four genes were induced in opr3 anthers within 0.5-1 h of JA treatment while the remaining genes were up-regulated only 1-8 h after JA application. None of these genes was induced by JA in anthers of the coil mutant that is deficient in JA responsiveness. The four early-induced genes in opr3 encode lipoxygenase, a putative bHLH transcription factor, epithiospecifier protein and an unknown protein. We propose that these and other early components may be involved in JA signaling and in the initiation of developmental processes. The four late genes encode an extensin-like protein, a peptide transporter and two unknown proteins, which may represent components required later in anther and pollen maturation. Transcript profiling has provided a successful approach to identify genes involved in anther and pollen maturation in Arabidopsis.

Plant Cation-Chloride Cotransporters (CCC): Evolutionary Origins and Functional Insights.

PubMed

Henderson, Sam W; Wege, Stefanie; Gilliham, Matthew

2018-02-06

Genomes of unicellular and multicellular green algae, mosses, grasses and dicots harbor genes encoding cation-chloride cotransporters (CCC). CCC proteins from the plant kingdom have been comparatively less well investigated than their animal counterparts, but proteins from both plants and animals have been shown to mediate ion fluxes, and are involved in regulation of osmotic processes. In this review, we show that CCC proteins from plants form two distinct phylogenetic clades (CCC1 and CCC2). Some lycophytes and bryophytes possess members from each clade, most land plants only have members of the CCC1 clade, and green algae possess only the CCC2 clade. It is currently unknown whether CCC1 and CCC2 proteins have similar or distinct functions, however they are both more closely related to animal KCC proteins compared to NKCCs. Existing heterologous expression systems that have been used to functionally characterize plant CCC proteins, namely yeast and Xenopus laevis oocytes, have limitations that are discussed. Studies from plants exposed to chemical inhibitors of animal CCC protein function are reviewed for their potential to discern CCC function in planta. Thus far, mutations in plant CCC genes have been evaluated only in two species of angiosperms, and such mutations cause a diverse array of phenotypes-seemingly more than could simply be explained by localized disruption of ion transport alone. We evaluate the putative roles of plant CCC proteins and suggest areas for future investigation.

Crystal structure of the YDR533c S. cerevisiae protein, a class II member of the Hsp31 family.

PubMed

Graille, Marc; Quevillon-Cheruel, Sophie; Leulliot, Nicolas; Zhou, Cong-Zhao; Li de la Sierra Gallay, Ines; Jacquamet, Lilian; Ferrer, Jean-Luc; Liger, Dominique; Poupon, Anne; Janin, Joel; van Tilbeurgh, Herman

2004-05-01

The ORF YDR533c from Saccharomyces cerevisiae codes for a 25.5 kDa protein of unknown biochemical function. Transcriptome analysis of yeast has shown that this gene is activated in response to various stress conditions together with proteins belonging to the heat shock family. In order to clarify its biochemical function, we determined the crystal structure of YDR533c to 1.85 A resolution by the single anomalous diffraction method. The protein possesses an alpha/beta hydrolase fold and a putative Cys-His-Glu catalytic triad common to a large enzyme family containing proteases, amidotransferases, lipases, and esterases. The protein has strong structural resemblance with the E. coli Hsp31 protein and the intracellular protease I from Pyrococcus horikoshii, which are considered class I and class III members of the Hsp31 family, respectively. Detailed structural analysis strongly suggests that the YDR533c protein crystal structure is the first one of a class II member of the Hsp31 family.

Crystal structure of PAV1-137: a protein from the virus PAV1 that infects Pyrococcus abyssi.

PubMed

Leulliot, N; Quevillon-Cheruel, S; Graille, M; Geslin, C; Flament, D; Le Romancer, M; van Tilbeurgh, H

2013-01-01

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four- α -helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Proteome map of Aspergillus nidulans during osmoadaptation.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2007-09-01

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.

Irreversible temperature gating in trpv1 sheds light on channel activation

PubMed Central

Sánchez-Moreno, Ana; Guevara-Hernández, Eduardo; Contreras-Cervera, Ricardo; Rangel-Yescas, Gisela; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara

2018-01-01

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening. PMID:29869983

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

PubMed Central

Moodley, Yoshan; Uhr, Markus; Stamer, Christiana; Vauterin, Marc; Suerbaum, Sebastian; Achtman, Mark

2010-01-01

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown. PMID:20808891

A global overview of the genetic and functional diversity in the Helicobacter pylori cag pathogenicity island.

PubMed

Olbermann, Patrick; Josenhans, Christine; Moodley, Yoshan; Uhr, Markus; Stamer, Christiana; Vauterin, Marc; Suerbaum, Sebastian; Achtman, Mark; Linz, Bodo

2010-08-19

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI-carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.

Probiotics and antibiotics change microbial diversity and decrease gut growth in neonatal pigs

USDA-ARS?s Scientific Manuscript database

Both probiotics and antibiotics are used as a therapy against infection and gut inflammatory disorders. The impact of these interventions on the gut microbiota, intestinal function and protein metabolism in preterm infants is unknown. We investigated the effect of antibiotics and probiotics on inte...

Shedding new light on opsin evolution

PubMed Central

Porter, Megan L.; Blasic, Joseph R.; Bok, Michael J.; Cameron, Evan G.; Pringle, Thomas; Cronin, Thomas W.; Robinson, Phyllis R.

2012-01-01

Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. As genomic data have appeared and rapidly expanded in quantity, it has become possible to analyse opsins that functionally and histologically are less well characterized, and thus to examine opsin evolution strictly from a genetic perspective. We have incorporated these new data into a large-scale, genome-based analysis of opsin evolution. We use an extensive phylogeny of currently known opsin sequence diversity as a foundation for examining the evolutionary distributions of key functional features within the opsin clade. This new analysis illustrates the lability of opsin protein-expression patterns, site-specific functionality (i.e. counterion position) and G-protein binding interactions. Further, it demonstrates the limitations of current model organisms, and highlights the need for further characterization of many of the opsin sequence groups with unknown function. PMID:22012981

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae*

PubMed Central

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; Shirasaki, Dyna I.; Wang, Charles; Blaby-Haas, Crysten E.; Merchant, Sabeeha S.; Loo, Joseph A.; Clarke, Catherine F.

2015-01-01

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11. PMID:25631044

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

DOE PAGES

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; ...

2015-01-28

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Mammalian Per-Arnt-Sim proteins in environmental adaptation.

PubMed

McIntosh, Brian E; Hogenesch, John B; Bradfield, Christopher A

2010-01-01

The Per-Arnt-Sim (PAS) domain is conserved across the kingdoms of life and found in an ever-growing list of proteins. This domain can bind to and sense endogenous or xenobiotic small molecules such as molecular oxygen, cellular metabolites, or polyaromatic hydrocarbons. Members of this family are often found in pathways that regulate responses to environmental change; in mammals these include the hypoxia, circadian, and dioxin response pathways. These pathways function in development and throughout life to regulate cellular, organ, and whole-organism adaptive responses. Remarkably, in the case of the clock, this adaptation includes anticipation of environmental change. In this review, we summarize the roles of PAS domain-containing proteins in mammals. We provide structural evidence that functionally classifies both known and unknown biological roles. Finally, we discuss the role of PAS proteins in anticipation of and adaptation to environmental change.

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE PAGES

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

2014-11-20

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lassila, JK; Bernstein, SL; Kinney, JN

Bacterial microconnpartnnents (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microconnpartnnent shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coll. These shells differ substantially from previously isolated shell systems in thatmore » they are considerably smaller and more homogeneous, with measured diameters of 39 2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments. (C) 2014 Elsevier Ltd. All rights reserved.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

The Paris-Sud yeast structural genomics pilot-project: from structure to function.

PubMed

Quevillon-Cheruel, Sophie; Liger, Dominique; Leulliot, Nicolas; Graille, Marc; Poupon, Anne; Li de La Sierra-Gallay, Inès; Zhou, Cong-Zhao; Collinet, Bruno; Janin, Joël; Van Tilbeurgh, Herman

2004-01-01

We present here the outlines and results from our yeast structural genomics (YSG) pilot-project. A lab-scale platform for the systematic production and structure determination is presented. In order to validate this approach, 250 non-membrane proteins of unknown structure were targeted. Strategies and final statistics are evaluated. We finally discuss the opportunity of structural genomics programs to contribute to functional biochemical annotation.

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

PubMed

Wang, Nan; Zhang, Yeting; Gedvilaite, Erika; Loh, Jui Wan; Lin, Timothy; Liu, Xiuping; Liu, Chang-Gong; Kumar, Dibyendu; Donnelly, Robert; Raymond, Kimiyo; Schuchman, Edward H; Sleat, David E; Lobel, Peter; Xing, Jinchuan

2017-11-01

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect. © 2017 Wiley Periodicals, Inc.

Finding undetected protein associations in cell signaling by belief propagation.

PubMed

Bailly-Bechet, M; Borgs, C; Braunstein, A; Chayes, J; Dagkessamanskaia, A; François, J-M; Zecchina, R

2011-01-11

External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High-throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein-protein interaction network and mRNA expression profiles. This inference problem can be mapped onto the problem of finding appropriate optimal connected subgraphs of a network defined by these datasets. The optimization procedure turns out to be computationally intractable in general. Here we present a new distributed algorithm for this task, inspired from statistical physics, and apply this scheme to alpha factor and drug perturbations data in yeast. We identify the role of the COS8 protein, a member of a gene family of previously unknown function, and validate the results by genetic experiments. The algorithm we present is specially suited for very large datasets, can run in parallel, and can be adapted to other problems in systems biology. On renowned benchmarks it outperforms other algorithms in the field.

The CASTOR proteins are arginine sensors for the mTORC1 pathway

PubMed Central

Chantranupong, Lynne; Scaria, Sonia M.; Saxton, Robert A.; Gygi, Melanie P.; Shen, Kuang; Wyant, Gregory A.; Wang, Tim; Harper, J. Wade; Gygi, Steven P.; Sabatini, David M.

2016-01-01

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

New Genes and Functional Innovation in Mammals

PubMed Central

Luis Villanueva-Cañas, José; Ruiz-Orera, Jorge; Agea, M. Isabel; Gallo, Maria; Andreu, David

2017-01-01

Abstract The birth of genes that encode new protein sequences is a major source of evolutionary innovation. However, we still understand relatively little about how these genes come into being and which functions they are selected for. To address these questions, we have obtained a large collection of mammalian-specific gene families that lack homologues in other eukaryotic groups. We have combined gene annotations and de novo transcript assemblies from 30 different mammalian species, obtaining ∼6,000 gene families. In general, the proteins in mammalian-specific gene families tend to be short and depleted in aromatic and negatively charged residues. Proteins which arose early in mammalian evolution include milk and skin polypeptides, immune response components, and proteins involved in reproduction. In contrast, the functions of proteins which have a more recent origin remain largely unknown, despite the fact that these proteins also have extensive proteomics support. We identify several previously described cases of genes originated de novo from noncoding genomic regions, supporting the idea that this mechanism frequently underlies the evolution of new protein-coding genes in mammals. Finally, we show that most young mammalian genes are preferentially expressed in testis, suggesting that sexual selection plays an important role in the emergence of new functional genes. PMID:28854603

iDBPs: a web server for the identification of DNA binding proteins

PubMed Central

Nimrod, Guy; Schushan, Maya; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2010-01-01

Summary: The iDBPs server uses the three-dimensional (3D) structure of a query protein to predict whether it binds DNA. First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence. We have trained and tested the classifier on various datasets and shown that it outperformed related methods. On a dataset that reflects the fraction of DNA binding proteins (DBPs) in a proteome, the area under the ROC curve was 0.90. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies. Availability: http://idbps.tau.ac.il/ Contact: NirB@tauex.tau.ac.il Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20089514

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

PubMed

Wu, Chenggang; Al Mamun, Abu Amar Mohamed; Luong, Truc Thanh; Hu, Bo; Gu, Jianhua; Lee, Ju Huck; D'Amore, Melissa; Das, Asis; Ton-That, Hung

2018-04-24

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the Δ ftsX and Δ envC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of Δ ftsX and Δ envC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated. IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum , due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the Δ ftsX and Δ envC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen. Copyright © 2018 Wu et al.

Genome-wide survey of DNA-binding proteins in Arabidopsis thaliana: analysis of distribution and functions.

PubMed

Malhotra, Sony; Sowdhamini, Ramanathan

2013-08-01

The interaction of proteins with their respective DNA targets is known to control many high-fidelity cellular processes. Performing a comprehensive survey of the sequenced genomes for DNA-binding proteins (DBPs) will help in understanding their distribution and the associated functions in a particular genome. Availability of fully sequenced genome of Arabidopsis thaliana enables the review of distribution of DBPs in this model plant genome. We used profiles of both structure and sequence-based DNA-binding families, derived from PDB and PFam databases, to perform the survey. This resulted in 4471 proteins, identified as DNA-binding in Arabidopsis genome, which are distributed across 300 different PFam families. Apart from several plant-specific DNA-binding families, certain RING fingers and leucine zippers also had high representation. Our search protocol helped to assign DNA-binding property to several proteins that were previously marked as unknown, putative or hypothetical in function. The distribution of Arabidopsis genes having a role in plant DNA repair were particularly studied and noted for their functional mapping. The functions observed to be overrepresented in the plant genome harbour DNA-3-methyladenine glycosylase activity, alkylbase DNA N-glycosylase activity and DNA-(apurinic or apyrimidinic site) lyase activity, suggesting their role in specialized functions such as gene regulation and DNA repair.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

PubMed

Morozov, Sergey Y; Milyutina, Irina A; Bobrova, Vera K; Ryazantsev, Dmitry Y; Erokhina, Tatiana N; Zavriev, Sergey K; Agranovsky, Alexey A; Solovyev, Andrey G; Troitsky, Alexey V

2015-12-01

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

A structural-alphabet-based strategy for finding structural motifs across protein families

PubMed Central

Wu, Chih Yuan; Chen, Yao Chi; Lim, Carmay

2010-01-01

Proteins with insignificant sequence and overall structure similarity may still share locally conserved contiguous structural segments; i.e. structural/3D motifs. Most methods for finding 3D motifs require a known motif to search for other similar structures or functionally/structurally crucial residues. Here, without requiring a query motif or essential residues, a fully automated method for discovering 3D motifs of various sizes across protein families with different folds based on a 16-letter structural alphabet is presented. It was applied to structurally non-redundant proteins bound to DNA, RNA, obligate/non-obligate proteins as well as free DNA-binding proteins (DBPs) and proteins with known structures but unknown function. Its usefulness was illustrated by analyzing the 3D motifs found in DBPs. A non-specific motif was found with a ‘corner’ architecture that confers a stable scaffold and enables diverse interactions, making it suitable for binding not only DNA but also RNA and proteins. Furthermore, DNA-specific motifs present ‘only’ in DBPs were discovered. The motifs found can provide useful guidelines in detecting binding sites and computational protein redesign. PMID:20525797

Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi*

PubMed Central

Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina

2017-01-01

Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types. PMID:27852749

Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded

PubMed Central

Yu, Jianzhong; Zheng, Yonggang; Dong, Jixin; Klusza, Stephen; Deng, Wu-Min; Pan, Duojia

2010-01-01

Summary The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra (Kbr) as another upstream component of the Hippo signaling pathway. We show that Kbr functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function, and implicate Kbr as a potential tumor suppressor with relevance to neurofibromatosis. PMID:20159598

Identification of a new EF-hand superfamily member from Trypanosoma brucei

NASA Technical Reports Server (NTRS)

Wong, S.; Kretsinger, R. H.; Campbell, D. A.

1992-01-01

We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

PubMed

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

A Framework for Globular Proteins

NASA Astrophysics Data System (ADS)

Lezon, Timothy

2006-03-01

Due to their remarkable chemical specificity and diversity, globular proteins play a crucial role in the network of molecular interactions of life. Over the past several decades, much experimental data has been accumulated on proteins, but the overarching principles that govern the general features of proteins remain largely unknown. Here, a novel framework for understanding many key attributes of globular proteins is presented. This framework suggests that the characteristics of globular proteins that make them well-suited for biological function are the emergent properties of a unique phase of matter. Implications of this picture include the provision of a fixed backdrop for molecular evolution and natural selection and design restrictions on molecular machinery. The work described here was carried out in collaboration with Jayanth Banavar and Amos Maritan.

The Enzyme Function Initiative†

PubMed Central

Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

2011-01-01

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID:21999478

The Enzyme Function Initiative.

PubMed

Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

2011-11-22

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts. © 2011 American Chemical Society

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027Wmore » revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Shanklin, John; Khusnutdinova, Anna

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae

PubMed Central

Specht, Sebastian; Miller, Stephanie B.M.

2011-01-01

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors. PMID:22065637

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE PAGES

Huang, Lili; Shanklin, John; Khusnutdinova, Anna; ...

2016-06-20

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Analysis of the relationships between evolvability, thermodynamics, and the functions of intrinsically disordered proteins/regions.

PubMed

Huang, He; Sarai, Akinori

2012-12-01

The evolvability of proteins is not only restricted by functional and structural importance, but also by other factors such as gene duplication, protein stability, and an organism's robustness. Recently, intrinsically disordered proteins (IDPs)/regions (IDRs) have been suggested to play a role in facilitating protein evolution. However, the mechanisms by which this occurs remain largely unknown. To address this, we have systematically analyzed the relationship between the evolvability, stability, and function of IDPs/IDRs. Evolutionary analysis shows that more recently emerged IDRs have higher evolutionary rates with more functional constraints relaxed (or experiencing more positive selection), and that this may have caused accelerated evolution in the flanking regions and in the whole protein. A systematic analysis of observed stability changes due to single amino acid mutations in IDRs and ordered regions shows that while most mutations induce a destabilizing effect in proteins, mutations in IDRs cause smaller stability changes than in ordered regions. The weaker impact of mutations in IDRs on protein stability may have advantages for protein evolvability in the gain of new functions. Interestingly, however, an analysis of functional motifs in the PROSITE and ELM databases showed that motifs in IDRs are more conserved, characterized by smaller entropy and lower evolutionary rate, than in ordered regions. This apparently opposing evolutionary effect may be partly due to the flexible nature of motifs in IDRs, which require some key amino acid residues to engage in tighter interactions with other molecules. Our study suggests that the unique conformational and thermodynamic characteristics of IDPs/IDRs play an important role in the evolvability of proteins to gain new functions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction.

PubMed

Bossi, Flavia; Fan, Jue; Xiao, Jun; Chandra, Lilyana; Shen, Max; Dorone, Yanniv; Wagner, Doris; Rhee, Seung Y

2017-06-26

The molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. To identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.

The Survival Motor Neuron Protein Forms Soluble Glycine Zipper Oligomers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, Renee; Gupta, Kushol; Ninan, Nisha S.

2012-11-01

The survival motor neuron (SMN) protein forms the oligomeric core of a multiprotein complex that functions in spliceosomal snRNP biogenesis. Loss of function mutations in the SMN gene cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Nearly half of the known SMA patient missense mutations map to the SMN YG-box, a highly conserved oligomerization domain of unknown structure that contains a (YxxG)3 motif. Here, we report that the SMN YG-box forms helical oligomers similar to the glycine zippers found in transmembrane channel proteins. A network of tyrosine-glycine packing between helices drives formation of soluble YG-box oligomers,more » providing a structural basis for understanding SMN oligomerization and for relating defects in oligomerization to the mutations found in SMA patients. These results have important implications for advancing our understanding of SMN function and glycine zipper-mediated helix-helix interactions.« less

A-to-I editing of coding and non-coding RNAs by ADARs

PubMed Central

Nishikura, Kazuko

2016-01-01

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference. PMID:26648264

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

Nucleophosmin/B23 regulates ubiquitin dynamics in nucleoli by recruiting deubiquitylating enzyme USP36.

PubMed

Endo, Akinori; Kitamura, Naomi; Komada, Masayuki

2009-10-09

The nucleolus is a subnuclear compartment with multiple cellular functions, including ribosome biogenesis. USP36 is a deubiquitylating enzyme that localizes to nucleoli and plays an essential role in regulating the structure and function of the organelle. However, how the localization of USP36 is regulated remains unknown. Here, we identified a short stretch of basic amino acids (RGKEKKIKKFKREKRR) that resides in the C-terminal region of USP36 and serves as a nucleolar localization signal for the protein. We found that this motif interacts with a central acidic region of nucleophosmin/B23, a major nucleolar protein involved in various nucleolar functions. Knockdown of nucleophosmin/B23 resulted in a significant reduction in the amount of USP36 in nucleoli, without affecting the cellular USP36 level. This was associated with elevated ubiquitylation levels of fibrillarin, a USP36 substrate protein in nucleoli. We conclude that nucleophosmin/B23 recruits USP36 to nucleoli, thereby serving as a platform for the regulation of nucleolar protein functions through ubiquitylation/deubiquitylation.

Recognition of functional sites in protein structures.

PubMed

Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

2004-06-04

Recognition of regions on the surface of one protein, that are similar to a binding site of another is crucial for the prediction of molecular interactions and for functional classifications. We first describe a novel method, SiteEngine, that assumes no sequence or fold similarities and is able to recognize proteins that have similar binding sites and may perform similar functions. We achieve high efficiency and speed by introducing a low-resolution surface representation via chemically important surface points, by hashing triangles of physico-chemical properties and by application of hierarchical scoring schemes for a thorough exploration of global and local similarities. We proceed to rigorously apply this method to functional site recognition in three possible ways: first, we search a given functional site on a large set of complete protein structures. Second, a potential functional site on a protein of interest is compared with known binding sites, to recognize similar features. Third, a complete protein structure is searched for the presence of an a priori unknown functional site, similar to known sites. Our method is robust and efficient enough to allow computationally demanding applications such as the first and the third. From the biological standpoint, the first application may identify secondary binding sites of drugs that may lead to side-effects. The third application finds new potential sites on the protein that may provide targets for drug design. Each of the three applications may aid in assigning a function and in classification of binding patterns. We highlight the advantages and disadvantages of each type of search, provide examples of large-scale searches of the entire Protein Data Base and make functional predictions.

Defining the Predicted Protein Secretome of the Fungal Wheat Leaf Pathogen Mycosphaerella graminicola

PubMed Central

Morais do Amaral, Alexandre; Antoniw, John; Rudd, Jason J.; Hammond-Kosack, Kim E.

2012-01-01

The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies. PMID:23236356

Redox proteomics of tomato in response to Pseudomonas syringae infection

PubMed Central

Balmant, Kelly Mayrink; Parker, Jennifer; Yoo, Mi-Jeong; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2015-01-01

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation–reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses. PMID:26504582

The cell envelope proteome of Aggregatibacter actinomycetemcomitans

PubMed Central

Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

2014-01-01

Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome.

PubMed

Kamal, Abu Hena Mostafa; Cho, Kun; Komatsu, Setsuko; Uozumi, Nobuyuki; Choi, Jong-Soon; Woo, Sun Hee

2012-05-01

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia.

PubMed

Xie, Bingxian; Chen, Qiaoli; Chen, Liang; Sheng, Yang; Wang, Hong Yu; Chen, Shuai

2016-11-01

The AS160 (Akt substrate of 160 kDa) is a Rab-GTPase activating protein (RabGAP) with several other functional domains, and its deficiency in mice or human patients lowers GLUT4 protein levels and causes severe insulin resistance. How its deficiency causes diminished GLUT4 proteins remains unknown. We found that the deletion of AS160 decreased GLUT4 levels in a cell/tissue-autonomous manner. Consequently, skeletal muscle-specific deletion of AS160 caused postprandial hyperglycemia and hyperinsulinemia. The pathogenic effects of AS160 deletion are mainly, if not exclusively, due to the loss of its RabGAP function since the RabGAP-inactive AS160 R917K mutant mice phenocopied the AS160 knockout mice. The inactivation of RabGAP of AS160 promotes lysosomal degradation of GLUT4, and the inhibition of lysosome function could restore GLUT4 protein levels. Collectively, these findings demonstrate that the RabGAP activity of AS160 maintains GLUT4 protein levels in a cell/tissue-autonomous manner and its inactivation causes lysosomal degradation of GLUT4 and postprandial hyperglycemia and hyperinsulinemia. © 2016 by the American Diabetes Association.

PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity.

PubMed

Feng, Baomin; Ma, Shisong; Chen, Sixue; Zhu, Ning; Zhang, Shuxin; Yu, Bin; Yu, Yu; Le, Brandon; Chen, Xuemei; Dinesh-Kumar, Savithramma P; Shan, Libo; He, Ping

2016-12-01

Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead-associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively. © 2016 The Authors.

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila.

PubMed

Barton, Lacy J; Wilmington, Shameika R; Martin, Melinda J; Skopec, Hannah M; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2014-06-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. Copyright © 2014 by the Genetics Society of America.

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

PubMed Central

Barton, Lacy J.; Wilmington, Shameika R.; Martin, Melinda J.; Skopec, Hannah M.; Lovander, Kaylee E.; Pinto, Belinda S.; Geyer, Pamela K.

2014-01-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. PMID:24700158

Shotgun protein sequencing: assembly of peptide tandem mass spectra from mixtures of modified proteins.

PubMed

Bandeira, Nuno; Clauser, Karl R; Pevzner, Pavel A

2007-07-01

Despite significant advances in the identification of known proteins, the analysis of unknown proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for sequencing of unknown proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA sequencing strategies have not been extended to automated protein sequencing. We demonstrate, for the first time, the feasibility of automated shotgun protein sequencing of protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various proteins in western diamondback rattlesnake venom. We further argue that shotgun protein sequencing has the potential to overcome the limitations of current protein sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown proteins.

Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury.

PubMed

Shah, Dilip; Romero, Freddy; Duong, Michelle; Wang, Nadan; Paudyal, Bishnuhari; Suratt, Benjamin T; Kallen, Caleb B; Sun, Jianxin; Zhu, Ying; Walsh, Kenneth; Summer, Ross

2015-06-12

Obesity is a risk factor for the development of acute respiratory distress syndrome (ARDS) but mechanisms mediating this association are unknown. While obesity is known to impair systemic blood vessel function, and predisposes to systemic vascular diseases, its effects on the pulmonary circulation are largely unknown. We hypothesized that the chronic low grade inflammation of obesity impairs pulmonary vascular homeostasis and primes the lung for acute injury. The lung endothelium from obese mice expressed higher levels of leukocyte adhesion markers and lower levels of cell-cell junctional proteins when compared to lean mice. We tested whether systemic factors are responsible for these alterations in the pulmonary endothelium; treatment of primary lung endothelial cells with obese serum enhanced the expression of adhesion proteins and reduced the expression of endothelial junctional proteins when compared to lean serum. Alterations in pulmonary endothelial cells observed in obese mice were associated with enhanced susceptibility to LPS-induced lung injury. Restoring serum adiponectin levels reversed the effects of obesity on the lung endothelium and attenuated susceptibility to acute injury. Our work indicates that obesity impairs pulmonary vascular homeostasis and enhances susceptibility to acute injury and provides mechanistic insight into the increased prevalence of ARDS in obese humans.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury

PubMed Central

Shah, Dilip; Romero, Freddy; Duong, Michelle; Wang, Nadan; Paudyal, Bishnuhari; Suratt, Benjamin T.; Kallen, Caleb B.; Sun, Jianxin; Zhu, Ying; Walsh, Kenneth; Summer, Ross

2015-01-01

Obesity is a risk factor for the development of acute respiratory distress syndrome (ARDS) but mechanisms mediating this association are unknown. While obesity is known to impair systemic blood vessel function, and predisposes to systemic vascular diseases, its effects on the pulmonary circulation are largely unknown. We hypothesized that the chronic low grade inflammation of obesity impairs pulmonary vascular homeostasis and primes the lung for acute injury. The lung endothelium from obese mice expressed higher levels of leukocyte adhesion markers and lower levels of cell-cell junctional proteins when compared to lean mice. We tested whether systemic factors are responsible for these alterations in the pulmonary endothelium; treatment of primary lung endothelial cells with obese serum enhanced the expression of adhesion proteins and reduced the expression of endothelial junctional proteins when compared to lean serum. Alterations in pulmonary endothelial cells observed in obese mice were associated with enhanced susceptibility to LPS-induced lung injury. Restoring serum adiponectin levels reversed the effects of obesity on the lung endothelium and attenuated susceptibility to acute injury. Our work indicates that obesity impairs pulmonary vascular homeostasis and enhances susceptibility to acute injury and provides mechanistic insight into the increased prevalence of ARDS in obese humans. PMID:26068229

LECTINPred: web Server that Uses Complex Networks of Protein Structure for Prediction of Lectins with Potential Use as Cancer Biomarkers or in Parasite Vaccine Design.

PubMed

Munteanu, Cristian R; Pedreira, Nieves; Dorado, Julián; Pazos, Alejandro; Pérez-Montoto, Lázaro G; Ubeira, Florencio M; González-Díaz, Humberto

2014-04-01

Lectins (Ls) play an important role in many diseases such as different types of cancer, parasitic infections and other diseases. Interestingly, the Protein Data Bank (PDB) contains +3000 protein 3D structures with unknown function. Thus, we can in principle, discover new Ls mining non-annotated structures from PDB or other sources. However, there are no general models to predict new biologically relevant Ls based on 3D chemical structures. We used the MARCH-INSIDE software to calculate the Markov-Shannon 3D electrostatic entropy parameters for the complex networks of protein structure of 2200 different protein 3D structures, including 1200 Ls. We have performed a Linear Discriminant Analysis (LDA) using these parameters as inputs in order to seek a new Quantitative Structure-Activity Relationship (QSAR) model, which is able to discriminate 3D structure of Ls from other proteins. We implemented this predictor in the web server named LECTINPred, freely available at http://bio-aims.udc.es/LECTINPred.php. This web server showed the following goodness-of-fit statistics: Sensitivity=96.7 % (for Ls), Specificity=87.6 % (non-active proteins), and Accuracy=92.5 % (for all proteins), considering altogether both the training and external prediction series. In mode 2, users can carry out an automatic retrieval of protein structures from PDB. We illustrated the use of this server, in operation mode 1, performing a data mining of PDB. We predicted Ls scores for +2000 proteins with unknown function and selected the top-scored ones as possible lectins. In operation mode 2, LECTINPred can also upload 3D structural models generated with structure-prediction tools like LOMETS or PHYRE2. The new Ls are expected to be of relevance as cancer biomarkers or useful in parasite vaccine design. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Approaches for assessing and discovering protein interactions in cancer

PubMed Central

Mohammed, Hisham; Carroll, Jason S.

2013-01-01

Significant insight into the function of proteins, can be delineated by discovering and characterising interacting proteins. There are numerous methods for the discovery of unknown associated protein networks, with purification of the bait (the protein of interest) followed by Mass Spectrometry (MS) as a common theme. In recent years, advances have permitted the purification of endogenous proteins and methods for scaling down starting material. As such, approaches for rapid, unbiased identification of protein interactomes are becoming a standard tool in the researchers toolbox, rather than a technique that is only available to specialists. This review will highlight some of the recent technical advances in proteomic based discovery approaches, the pros and cons of various methods and some of the key findings in cancer related systems. PMID:24072816

Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

PubMed Central

Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

2014-01-01

The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8. PMID:28250384

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

PubMed Central

He, Yi-Ming; Ma, Bin-Guang

2016-01-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

NASA Astrophysics Data System (ADS)

He, Yi-Ming; Ma, Bin-Guang

2016-05-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

Heterogeneity in the A33 Protein Impacts the Cross-Protective Efficacy of a Candidate Smallpox DNA Vaccine

DTIC Science & Technology

2008-01-01

include myocarditis, progressive vaccinia, eczema vaccinatum, and even death (Bray, 2003; Cassimatis et al., 2004; Eckart et al., 2004;Kretzschmaret...hundreds of genes, many of which encode for immunomodulatory molecules or molecules with unknown function . The safety risks posed by these molecules...1G10 or function indirectly by impacting the conformation of the physical epitope. However, findings in the literature support the conclusion that

The Functional Impact of Alternative Splicing in Cancer.

PubMed

Climente-González, Héctor; Porta-Pardo, Eduard; Godzik, Adam; Eyras, Eduardo

2017-08-29

Alternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remain mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein-protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer, and some of them could potentially be considered alternative splicing drivers (AS drivers). Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Identification of significantly mutated regions across cancer types highlights a rich landscape of functional molecular alterations

PubMed Central

Araya, Carlos L.; Cenik, Can; Reuter, Jason A.; Kiss, Gert; Pande, Vijay S.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

Cancer sequencing studies have primarily identified cancer-driver genes by the accumulation of protein-altering mutations. An improved method would be annotation-independent, sensitive to unknown distributions of functions within proteins, and inclusive of non-coding drivers. We employed density-based clustering methods in 21 tumor types to detect variably-sized significantly mutated regions (SMRs). SMRs reveal recurrent alterations across a spectrum of coding and non-coding elements, including transcription factor binding sites and untranslated regions mutated in up to ∼15% of specific tumor types. SMRs reveal spatial clustering of mutations at molecular domains and interfaces, often with associated changes in signaling. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated among tumor types, as exemplified by a linker region of PIK3CA in which biophysical simulations suggest mutations affect regulatory interactions. The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of functionally-agnostic driver identification. PMID:26691984

Concentration-dependent Cu(II) binding to prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

2008-03-01

The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

Sox proteins in melanocyte development and melanoma

PubMed Central

Harris, Melissa L.; Baxter, Laura L.; Loftus, Stacie K.; Pavan, William J.

2010-01-01

Over ten years has passed since the first Sox gene was implicated in melanocyte development. Since then, we have discovered that SOX5, SOX9, SOX10 and SOX18 all participate as transcription factors that affect key melanocytic genes in both regulatory and modulatory fashions. Both SOX9 and SOX10 play major roles in the establishment and normal function of the melanocyte; SOX10 has been shown to heavily influence melanocyte development and SOX9 has been implicated in melanogenesis in the adult. Despite these advances, the precise cellular and molecular details of how these SOX proteins are regulated and interact during all stages of the melanocyte life cycle remain unknown. Improper regulation of SOX9 or SOX10 is also associated with cancerous transformation, and thus understanding the normal function of SOX proteins in the melanocyte will be key to revealing how these proteins contribute to melanoma. PMID:20444197

Complete fold annotation of the human proteome using a novel structural feature space.

PubMed

Middleton, Sarah A; Illuminati, Joseph; Kim, Junhyong

2017-04-13

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this methodmore » by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Finally, our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.« less

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa

DOE PAGES

Welner, Ditte Hededam; Tsai, Alex Yi-Lin; DeGiovanni, Andy M.; ...

2017-03-29

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstratemore » DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.« less

Complete fold annotation of the human proteome using a novel structural feature space

PubMed Central

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

2017-01-01

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families. PMID:28406174

Plant Cation-Chloride Cotransporters (CCC): Evolutionary Origins and Functional Insights

PubMed Central

Gilliham, Matthew

2018-01-01

Genomes of unicellular and multicellular green algae, mosses, grasses and dicots harbor genes encoding cation-chloride cotransporters (CCC). CCC proteins from the plant kingdom have been comparatively less well investigated than their animal counterparts, but proteins from both plants and animals have been shown to mediate ion fluxes, and are involved in regulation of osmotic processes. In this review, we show that CCC proteins from plants form two distinct phylogenetic clades (CCC1 and CCC2). Some lycophytes and bryophytes possess members from each clade, most land plants only have members of the CCC1 clade, and green algae possess only the CCC2 clade. It is currently unknown whether CCC1 and CCC2 proteins have similar or distinct functions, however they are both more closely related to animal KCC proteins compared to NKCCs. Existing heterologous expression systems that have been used to functionally characterize plant CCC proteins, namely yeast and Xenopus laevis oocytes, have limitations that are discussed. Studies from plants exposed to chemical inhibitors of animal CCC protein function are reviewed for their potential to discern CCC function in planta. Thus far, mutations in plant CCC genes have been evaluated only in two species of angiosperms, and such mutations cause a diverse array of phenotypes—seemingly more than could simply be explained by localized disruption of ion transport alone. We evaluate the putative roles of plant CCC proteins and suggest areas for future investigation. PMID:29415511

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

PubMed

Bukiya, Anna N; Durdagi, Serdar; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-04-14

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families

PubMed Central

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Švedas, Vytas

2014-01-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure–function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. PMID:24852248

Functional proteomics of nonalcoholic steatohepatitis: Mitochondrial proteins as targets of S-adenosylmethionine

PubMed Central

Santamaría, Enrique; Avila, Matías A.; Latasa, M. Ujue; Rubio, Angel; Martín-Duce, Antonio; Lu, Shelly C.; Mato, José M.; Corrales, Fernando J.

2003-01-01

Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A−/−) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A−/− expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at ≈8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase β-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in ob/ob mice and obese patients who are at risk for nonalcoholic steatohepatitis. PMID:12631701

TAL effectors and the executor R genes

PubMed Central

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized—recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance. PMID:26347759

TAL effectors and the executor R genes.

PubMed

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized-recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro *

PubMed Central

Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

2016-01-01

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

Irreversible temperature gating in trpv1 sheds light on channel activation.

PubMed

Sánchez-Moreno, Ana; Guevara-Hernández, Eduardo; Contreras-Cervera, Ricardo; Rangel-Yescas, Gisela; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara; Islas, León D

2018-06-05

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening. © 2018, Sánchez-Moreno et al.

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry

PubMed Central

Wang, Da-Zhi; Dong, Hong-Po; Li, Cheng; Xie, Zhang-Xian; Lin, Lin; Hong, Hua-Sheng

2011-01-01

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions. PMID:21904561

Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway.

PubMed

Lin, Li; Hales, Chadwick M; Garber, Kathryn; Jin, Peng

2014-06-15

Polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. FTO is a nuclear protein and its physiological function remains largely unknown, but alterations in its expression in mice influence energy expenditure, food intake and, ultimately, body weight. To understand the molecular functions of FTO, we performed a yeast two-hybrid screen to identify the protein(s) that could directly interact with human FTO protein. Using multiple assays, we demonstrate that FTO interacts with three isoforms of calcium/calmodulin-dependent protein kinase II: α, β and γ, which are protein kinases that phosphorylate a broad range of substrates. This interaction is functional; overexpression of FTO delays the dephosphorylation of cAMP response element-binding protein (CREB) in human neuroblastoma (SK-N-SH) cells, which in turn leads to a dramatic increase in the expression of the CREB targets neuropeptide receptor 1 (NPY1R) and brain-derived neurotrophic factor (BDNF), which already are known to regulate food intake and energy homeostasis. Thus, our results suggest that FTO could modulate obesity by regulating the activity of the CREB signaling pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of the heat shock protein HSP27 on androgen receptor expression and function in prostate cancer cells.

PubMed

Stope, Matthias B; Schubert, Tina; Staar, Doreen; Rönnau, Cindy; Streitbörger, Andreas; Kroeger, Nils; Kubisch, Constanze; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

2012-06-01

Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.

Ab Initio Structural Modeling of and Experimental Validation for Chlamydia trachomatis Protein CT296 Reveal Structural Similarity to Fe(II) 2-Oxoglutarate-Dependent Enzymes▿

PubMed Central

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott; Battaile, Kevin P.; Zhang, Yang; Hefty, P. Scott

2011-01-01

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF) CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-Å Cα root mean square deviation [RMSD]) the high-resolution (1.8-Å) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur. PMID:21965559

Ab initio structural modeling of and experimental validation for Chlamydia trachomatis protein CT296 reveal structural similarity to Fe(II) 2-oxoglutarate-dependent enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott

2012-02-13

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF)more » CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-{angstrom} C{alpha} root mean square deviation [RMSD]) the high-resolution (1.8-{angstrom}) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur.« less

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: a structural biology web resource.

PubMed

Gifford, Lida K; Carter, Lester G; Gabanyi, Margaret J; Berman, Helen M; Adams, Paul D

2012-06-01

The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/ ) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB ( http://sbkb.org ), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a functional sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.

Functions of IQD proteins as hubs in cellular calcium and auxin signaling: A toolbox for shape formation and tissue-specification in plants?

PubMed

Bürstenbinder, Katharina; Mitra, Dipannita; Quegwer, Jakob

2017-06-03

Calcium (Ca 2+ ) ions play pivotal roles as second messengers in intracellular signal transduction, and coordinate many biological processes. Changes in intracellular Ca 2+ levels are perceived by Ca 2+ sensors such as calmodulin (CaM) and CaM-like (CML) proteins, which transduce Ca 2+ signals into cellular responses by regulation of diverse target proteins. Insights into molecular functions of CaM targets are thus essential to understand the molecular and cellular basis of Ca 2+ signaling. During the last decade, IQ67-domain (IQD) proteins emerged as the largest class of CaM targets in plants with mostly unknown functions. In the March issue of Plant Physiology, we presented the first comprehensive characterization of the 33-membered IQD family in Arabidopsis thaliana. We showed, by analysis of the subcellular localization of translational green fluorescent protein (GFP) fusion proteins, that most IQD members label microtubules (MTs), and additionally often localize to the cell nucleus or to membranes, where they recruit CaM Ca 2+ sensors. Important functions at MTs are supported by altered MT organization and plant growth in IQD gain-of-function lines. Because IQD proteins share structural hallmarks of scaffold proteins, we propose roles of IQDs in the assembly of macromolecular complexes to orchestrate Ca 2+ CaM signaling from membranes to the nucleus. Interestingly, expression of several IQDs is regulated by auxin, which suggests functions of IQDs as hubs in cellular auxin and calcium signaling to regulate plant growth and development.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

PubMed

Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

2015-06-01

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes.

PubMed

Antonenkov, Vasily D; Ohlmeier, Steffen; Sormunen, Raija T; Hiltunen, J Kalervo

2007-05-25

Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism.

Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

DOE PAGES

Natarajan, Padmaja; Punta, Marco; Kumar, Abhinav; ...

2015-01-16

N-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.

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. Recently it was found that, as in the previous case, the proteins have a structure unknown among modern enzymes. In this case, in vitro evolution started from a small, non-enzymatic protein. A similar selection process initiated from a library of random polypeptides is in progress. These results not only allow for estimating the occurrence of function in random protein assemblies but also provide evidence for the possibility of alternative protein worlds. Extant proteins might simply represent a frozen accident in the world of possible proteins. Alternative collections of proteins, even with similar functions, could originate alternative evolutionary paths.

Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

PubMed Central

Moller-Tank, Sven; Albritton, Lorraine M.; Rennert, Paul D.

2014-01-01

ABSTRACT T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs. PMID:24696470

Transcriptome and proteome analysis of ovaries of arrhenotokous and thelytokous Venturia canescens.

PubMed

Mateo Leach, I; Hesseling, A; Huibers, W H C; Witsenboer, H; Beukeboom, L W; van de Zande, L

2009-08-01

Under arrhenotoky, unfertilized haploid eggs develop as males but under thelytoky they develop into diploid females after they have undergone diploidy restoration. In the parasitoid wasp Venturia canescens both reproductive modes occur. Thelytoky is genetically determined but the underlying genetics of diploidy restoration remain unknown. In this study we aim to identify the genes and/or proteins that control thelytoky. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of total ovarian RNA and two-dimensional protein electrophoresis in combination with mass spectrometry revealed putative transcripts and proteins involved in arrhenotokous and thelytokous development. The detected tubulin and actin protein differences are most likely functionally related to the two types of reproduction.

Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori.

PubMed

Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

2016-04-22

The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study.

Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori

PubMed Central

Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

2016-01-01

The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography–tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study. PMID:27102218

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

PubMed

Huang, Sheng Yu; Chen, Sung Fang; Chen, Chun Hao; Huang, Hsuan Wei; Wu, Wen Guey; Sung, Wang Chou

2014-09-02

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals

PubMed Central

Taglialegna, Agustina; Navarro, Susanna; Ventura, Salvador; Garnett, James A.; Matthews, Steve; Penades, José R.; Lasa, Iñigo; Valle, Jaione

2016-01-01

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria. PMID:27327765

Lack of Spartin Protein in Troyer Syndrome

PubMed Central

Bakowska, Joanna C.; Wang, Heng; Xin, Baozhong; Sumner, Charlotte J.; Blackstone, Craig

2017-01-01

Background Hereditary spastic paraplegias (SPG1-SPG33) are characterized by progressive spastic weakness of the lower limbs. A nucleotide deletion (1110delA) in the (SPG20; OMIM 275900) spartin gene is the origin of autosomal recessive Troyer syndrome. This mutation is predicted to cause premature termination of the spartin protein. However, it remains unknown whether this truncated spartin protein is absent or is present and partially functional in patients. Objective To determine whether the truncated spartin protein is present or absent in cells derived from patients with Troyer syndrome. Design Case report. Setting Academic research. Patients We describe a new family with Troyer syndrome due to the 1110delA mutation. Main Outcome Measures We cultured primary fibroblasts and generated lymphoblasts from affected individuals, carriers, and control subjects and subjected these cells to immunoblot analyses. Results Spartin protein is undetectable in several cell lines derived from patients with Troyer syndrome. Conclusions Our data suggest that Troyer syndrome results from complete loss of spartin protein rather than from the predicted partly functional fragment. This may reflect increased protein degradation or impaired translation. PMID:18413476

Mining sequential patterns for protein fold recognition.

PubMed

Exarchos, Themis P; Papaloukas, Costas; Lampros, Christos; Fotiadis, Dimitrios I

2008-02-01

Protein data contain discriminative patterns that can be used in many beneficial applications if they are defined correctly. In this work sequential pattern mining (SPM) is utilized for sequence-based fold recognition. Protein classification in terms of fold recognition plays an important role in computational protein analysis, since it can contribute to the determination of the function of a protein whose structure is unknown. Specifically, one of the most efficient SPM algorithms, cSPADE, is employed for the analysis of protein sequence. A classifier uses the extracted sequential patterns to classify proteins in the appropriate fold category. For training and evaluating the proposed method we used the protein sequences from the Protein Data Bank and the annotation of the SCOP database. The method exhibited an overall accuracy of 25% in a classification problem with 36 candidate categories. The classification performance reaches up to 56% when the five most probable protein folds are considered.

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Hongbing; Franz, Carl J.; Wu, Guang

2014-02-15

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated intomore » Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.« less

Considerations to improve functional annotations in biological databases.

PubMed

Benítez-Páez, Alfonso

2009-12-01

Despite the great effort to design efficient systems allowing the electronic indexation of information concerning genes, proteins, structures, and interactions published daily in scientific journals, some problems are still observed in specific tasks such as functional annotation. The annotation of function is a critical issue for bioinformatic routines, such as for instance, in functional genomics and the further prediction of unknown protein function, which are highly dependent of the quality of existing annotations. Some information management systems evolve to efficiently incorporate information from large-scale projects, but often, annotation of single records from the literature is difficult and slow. In this short report, functional characterizations of a representative sample of the entire set of uncharacterized proteins from Escherichia coli K12 was compiled from Swiss-Prot, PubMed, and EcoCyc and demonstrate a functional annotation deficit in biological databases. Some issues are postulated as causes of the lack of annotation, and different solutions are evaluated and proposed to avoid them. The hope is that as a consequence of these observations, there will be new impetus to improve the speed and quality of functional annotation and ultimately provide updated, reliable information to the scientific community.

Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3.

PubMed

Wang, Yifan; Fang, Rui; Yuan, Yuan; Pan, Ming; Hu, Min; Zhou, Yanqin; Shen, Bang; Zhao, Junlong

2016-07-01

As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.

Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

PubMed

Dewhurst, Henry M; Choudhury, Shilpa; Torres, Matthew P

2015-08-01

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)--a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits--conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68

PubMed Central

Feracci, Mikael; Foot, Jaelle N.; Grellscheid, Sushma N.; Danilenko, Marina; Stehle, Ralf; Gonchar, Oksana; Kang, Hyun-Seo; Dalgliesh, Caroline; Meyer, N. Helge; Liu, Yilei; Lahat, Albert; Sattler, Michael; Eperon, Ian C.; Elliott, David J.; Dominguez, Cyril

2016-01-01

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome. PMID:26758068

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68.

PubMed

Feracci, Mikael; Foot, Jaelle N; Grellscheid, Sushma N; Danilenko, Marina; Stehle, Ralf; Gonchar, Oksana; Kang, Hyun-Seo; Dalgliesh, Caroline; Meyer, N Helge; Liu, Yilei; Lahat, Albert; Sattler, Michael; Eperon, Ian C; Elliott, David J; Dominguez, Cyril

2016-01-13

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.

The protein interaction map of bacteriophage lambda

PubMed Central

2011-01-01

Background Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. PMID:21943085

PaperBLAST: Text Mining Papers for Information about Homologs.

PubMed

Price, Morgan N; Arkin, Adam P

2017-01-01

Large-scale genome sequencing has identified millions of protein-coding genes whose function is unknown. Many of these proteins are similar to characterized proteins from other organisms, but much of this information is missing from annotation databases and is hidden in the scientific literature. To make this information accessible, PaperBLAST uses EuropePMC to search the full text of scientific articles for references to genes. PaperBLAST also takes advantage of curated resources (Swiss-Prot, GeneRIF, and EcoCyc) that link protein sequences to scientific articles. PaperBLAST's database includes over 700,000 scientific articles that mention over 400,000 different proteins. Given a protein of interest, PaperBLAST quickly finds similar proteins that are discussed in the literature and presents snippets of text from relevant articles or from the curators. PaperBLAST is available at http://papers.genomics.lbl.gov/. IMPORTANCE With the recent explosion of genome sequencing data, there are now millions of uncharacterized proteins. If a scientist becomes interested in one of these proteins, it can be very difficult to find information as to its likely function. Often a protein whose sequence is similar, and which is likely to have a similar function, has been studied already, but this information is not available in any database. To help find articles about similar proteins, PaperBLAST searches the full text of scientific articles for protein identifiers or gene identifiers, and it links these articles to protein sequences. Then, given a protein of interest, it can quickly find similar proteins in its database by using standard software (BLAST), and it can show snippets of text from relevant papers. We hope that PaperBLAST will make it easier for biologists to predict proteins' functions.

The Genome of S-PM2, a “Photosynthetic” T4-Type Bacteriophage That Infects Marine Synechococcus Strains

PubMed Central

Mann, Nicholas H.; Clokie, Martha R. J.; Millard, Andrew; Cook, Annabel; Wilson, William H.; Wheatley, Peter J.; Letarov, Andrey; Krisch, H. M.

2005-01-01

Bacteriophage S-PM2 infects several strains of the abundant and ecologically important marine cyanobacterium Synechococcus. A large lytic phage with an isometric icosahedral head, S-PM2 has a contractile tail and by this criterion is classified as a myovirus (1). The linear, circularly permuted, 196,280-bp double-stranded DNA genome of S-PM2 contains 37.8% G+C residues. It encodes 239 open reading frames (ORFs) and 25 tRNAs. Of these ORFs, 19 appear to encode proteins associated with the cell envelope, including a putative S-layer-associated protein. Twenty additional S-PM2 ORFs have homologues in the genomes of their cyanobacterial hosts. There is a group I self-splicing intron within the gene encoding the D1 protein. A total of 40 ORFs, organized into discrete clusters, encode homologues of T4 proteins involved in virion morphogenesis, nucleotide metabolism, gene regulation, and DNA replication and repair. The S-PM2 genome encodes a few surprisingly large (e.g., 3,779 amino acids) ORFs of unknown function. Our analysis of the S-PM2 genome suggests that many of the unknown S-PM2 functions may be involved in the adaptation of the metabolism of the host cell to the requirements of phage infection. This hypothesis originates from the identification of multiple phage-mediated modifications of the host's photosynthetic apparatus that appear to be essential for maintaining energy production during the lytic cycle. PMID:15838046

New TFII-I family target genes involved in embryonic development.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2009-09-04

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

New Genes and Functional Innovation in Mammals.

PubMed

Luis Villanueva-Cañas, José; Ruiz-Orera, Jorge; Agea, M Isabel; Gallo, Maria; Andreu, David; Albà, M Mar

2017-07-01

The birth of genes that encode new protein sequences is a major source of evolutionary innovation. However, we still understand relatively little about how these genes come into being and which functions they are selected for. To address these questions, we have obtained a large collection of mammalian-specific gene families that lack homologues in other eukaryotic groups. We have combined gene annotations and de novo transcript assemblies from 30 different mammalian species, obtaining ∼6,000 gene families. In general, the proteins in mammalian-specific gene families tend to be short and depleted in aromatic and negatively charged residues. Proteins which arose early in mammalian evolution include milk and skin polypeptides, immune response components, and proteins involved in reproduction. In contrast, the functions of proteins which have a more recent origin remain largely unknown, despite the fact that these proteins also have extensive proteomics support. We identify several previously described cases of genes originated de novo from noncoding genomic regions, supporting the idea that this mechanism frequently underlies the evolution of new protein-coding genes in mammals. Finally, we show that most young mammalian genes are preferentially expressed in testis, suggesting that sexual selection plays an important role in the emergence of new functional genes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

PubMed Central

Zylka, Mark J.; Sowa, Nathaniel A.; Taylor-Blake, Bonnie; Twomey, Margaret A.; Herrala, Annakaisa; Voikar, Vootele; Vihko, Pirkko

2008-01-01

SUMMARY Thiamine monophosphatase (TMPase, also known as Fluoride-Resistant Acid Phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of Prostatic Acid Phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent anti-nociceptive, anti-hyperalgesic and anti-allodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5’-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. PMID:18940592

Isolation, sequence analysis, and comparison of two plasmids (28 and 29 kilobases) from the biomining bacterium Leptospirillum ferrooxidans ATCC 49879.

PubMed

Coram, Nicolette J; van Zyl, Leonardo J; Rawlings, Douglas E

2005-11-01

Two plasmids, of 28,878 bp and 28,012 bp, were isolated from Leptospirillum ferrooxidans ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus Leptospirillum, is presented.

A novel salt-inducible gene SbSI-1 from Salicornia brachiata confers salt and desiccation tolerance in E. coli.

PubMed

Yadav, Narendra Singh; Rashmi, Deo; Singh, Dinkar; Agarwal, Pradeep K; Jha, Bhavanath

2012-02-01

Salicornia brachiata is one of the extreme salt tolerant plants and grows luxuriantly in coastal areas. Previously we have reported isolation and characterization of ESTs from S. brachiata with large number of unknown gene sequences. Reverse Northern analysis showed upregulation and downregulation of few unknown genes in response to salinity. Some of these unknown genes were made full length and their functional analysis is being tested. In this study, we have selected a novel unknown salt inducible gene SbSI-1 (Salicornia brachiata salt inducible-1) for the functional validation. The SbSI-1 (Gen-Bank accession number JF 965339) was made full length and characterized in detail for its functional validation under desiccation and salinity. The SbSI-1 gene is 917 bp long, and contained 437 bp 3' UTR, and 480 bp ORF region encoding 159 amino acids protein with estimated molecular mass of 18.39 kDa and pI 8.58. The real time PCR analysis revealed high transcript expression in salt, desiccation, cold and heat stresses. However, the maximum expression was obtained by desiccation. The ORF region of SbSI-1 was cloned in pET28a vector and transformed in BL21 (DE3) E. coli cells. The SbSI-1 recombinant E. coli cells showed tolerance to desiccation and salinity stress compared to only vector in the presence of stress.

Computational mining for hypothetical patterns of amino acid side chains in protein data bank (PDB)

NASA Astrophysics Data System (ADS)

Ghani, Nur Syatila Ab; Firdaus-Raih, Mohd

2018-04-01

The three-dimensional structure of a protein can provide insights regarding its function. Functional relationship between proteins can be inferred from fold and sequence similarities. In certain cases, sequence or fold comparison fails to conclude homology between proteins with similar mechanism. Since the structure is more conserved than the sequence, a constellation of functional residues can be similarly arranged among proteins of similar mechanism. Local structural similarity searches are able to detect such constellation of amino acids among distinct proteins, which can be useful to annotate proteins of unknown function. Detection of such patterns of amino acids on a large scale can increase the repertoire of important 3D motifs since available known 3D motifs currently, could not compensate the ever-increasing numbers of uncharacterized proteins to be annotated. Here, a computational platform for an automated detection of 3D motifs is described. A fuzzy-pattern searching algorithm derived from IMagine an Amino Acid 3D Arrangement search EnGINE (IMAAAGINE) was implemented to develop an automated method for searching of hypothetical patterns of amino acid side chains in Protein Data Bank (PDB), without the need for prior knowledge on related sequence or structure of pattern of interest. We present an example of the searches, which is the detection of a hypothetical pattern derived from known structural motif of C2H2 structural pattern from zinc fingers. The conservation of particular patterns of amino acid side chains in unrelated proteins is highlighted. This approach can act as a complementary method for available structure- and sequence-based platforms and may contribute in improving functional association between proteins.

Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

PubMed Central

Covian, Raul

2012-01-01

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria. PMID:22886415

PaperBLAST: Text Mining Papers for Information about Homologs

DOE PAGES

Price, Morgan N.; Arkin, Adam P.

2017-08-15

Large-scale genome sequencing has identified millions of protein-coding genes whose function is unknown. Many of these proteins are similar to characterized proteins from other organisms, but much of this information is missing from annotation databases and is hidden in the scientific literature. To make this information accessible, PaperBLAST uses EuropePMC to search the full text of scientific articles for references to genes. PaperBLAST also takes advantage of curated resources (Swiss-Prot, GeneRIF, and EcoCyc) that link protein sequences to scientific articles. PaperBLAST’s database includes over 700,000 scientific articles that mention over 400,000 different proteins. Given a protein of interest, PaperBLAST quicklymore » finds similar proteins that are discussed in the literature and presents snippets of text from relevant articles or from the curators. With the recent explosion of genome sequencing data, there are now millions of uncharacterized proteins. If a scientist becomes interested in one of these proteins, it can be very difficult to find information as to its likely function. Often a protein whose sequence is similar, and which is likely to have a similar function, has been studied already, but this information is not available in any database. To help find articles about similar proteins, PaperBLAST searches the full text of scientific articles for protein identifiers or gene identifiers, and it links these articles to protein sequences. Then, given a protein of interest, it can quickly find similar proteins in its database by using standard software (BLAST), and it can show snippets of text from relevant papers. We hope that PaperBLAST will make it easier for biologists to predict proteins’ functions.« less

PaperBLAST: Text Mining Papers for Information about Homologs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Arkin, Adam P.

Large-scale genome sequencing has identified millions of protein-coding genes whose function is unknown. Many of these proteins are similar to characterized proteins from other organisms, but much of this information is missing from annotation databases and is hidden in the scientific literature. To make this information accessible, PaperBLAST uses EuropePMC to search the full text of scientific articles for references to genes. PaperBLAST also takes advantage of curated resources (Swiss-Prot, GeneRIF, and EcoCyc) that link protein sequences to scientific articles. PaperBLAST’s database includes over 700,000 scientific articles that mention over 400,000 different proteins. Given a protein of interest, PaperBLAST quicklymore » finds similar proteins that are discussed in the literature and presents snippets of text from relevant articles or from the curators. With the recent explosion of genome sequencing data, there are now millions of uncharacterized proteins. If a scientist becomes interested in one of these proteins, it can be very difficult to find information as to its likely function. Often a protein whose sequence is similar, and which is likely to have a similar function, has been studied already, but this information is not available in any database. To help find articles about similar proteins, PaperBLAST searches the full text of scientific articles for protein identifiers or gene identifiers, and it links these articles to protein sequences. Then, given a protein of interest, it can quickly find similar proteins in its database by using standard software (BLAST), and it can show snippets of text from relevant papers. We hope that PaperBLAST will make it easier for biologists to predict proteins’ functions.« less

PaperBLAST: Text Mining Papers for Information about Homologs

PubMed Central

Arkin, Adam P.

2017-01-01

ABSTRACT Large-scale genome sequencing has identified millions of protein-coding genes whose function is unknown. Many of these proteins are similar to characterized proteins from other organisms, but much of this information is missing from annotation databases and is hidden in the scientific literature. To make this information accessible, PaperBLAST uses EuropePMC to search the full text of scientific articles for references to genes. PaperBLAST also takes advantage of curated resources (Swiss-Prot, GeneRIF, and EcoCyc) that link protein sequences to scientific articles. PaperBLAST’s database includes over 700,000 scientific articles that mention over 400,000 different proteins. Given a protein of interest, PaperBLAST quickly finds similar proteins that are discussed in the literature and presents snippets of text from relevant articles or from the curators. PaperBLAST is available at http://papers.genomics.lbl.gov/. IMPORTANCE With the recent explosion of genome sequencing data, there are now millions of uncharacterized proteins. If a scientist becomes interested in one of these proteins, it can be very difficult to find information as to its likely function. Often a protein whose sequence is similar, and which is likely to have a similar function, has been studied already, but this information is not available in any database. To help find articles about similar proteins, PaperBLAST searches the full text of scientific articles for protein identifiers or gene identifiers, and it links these articles to protein sequences. Then, given a protein of interest, it can quickly find similar proteins in its database by using standard software (BLAST), and it can show snippets of text from relevant papers. We hope that PaperBLAST will make it easier for biologists to predict proteins’ functions. PMID:28845458

Livedoid vasculopathy in a patient with factor V mutation (Leiden).

PubMed

Biedermann, T; Flaig, M J; Sander, C A

2000-09-01

Frequently, no underlying disease can be detected in patients with livedoid vasculopathy. For these forms, an unknown vaso-occlusive or thrombogenic process has been accused to play a role. Thus, a patient with livedoid vasculopathy was examined for different parameters which can be involved in coagulopathies. Laboratory studies for different autoantigen reactive immunoglobulins, cryoglobulins, and circulating immune complexes were carried out. Besides dermatopathologic examination, a biopsy specimen was analyzed by direct immunofluorescence for immunoglobulin (Ig) and complement deposits. Furthermore, hemostaseological function tests including activated protein C (APC) resistance were undertaken. Positive only at very low titres were antinuclear antibodies and c-ANCA, all other parameters were within normal ranges or negative. Direct immunofluorescence revealed IgM, C3 and fibrogen deposits. Hemostaseological function tests demonstrated a pathologic activated protein c resistance and PCR analysis a heterozygous defect of the factor V (Leiden). The diagnosis of livedoid vasculopathy associated with factor V mutation (Leiden) was made. Since the underlying cause for livedoid vasculopathy often remains unknown, we suggest that hemostaseological function tests including APC resistance and factor V gene mutation analysis should be carried out. Further studies have to follow in order to elucidate the role of mutant factor V in livedoid vasculopathy and in cutaneous ulcerations.

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

Higher plasma vitamin D binding protein and lower free calcitriol with tenofovir treatment: Cause of a functional vitamin D deficiency?

USDA-ARS?s Scientific Manuscript database

Background: Tenofovir disoproxil fumarate (TDF) causes bone, endocrine and renal changes, by unknown mechanism(s). There are limited data on tenofovir (TFV) pharmacokinetics and these effects. Methods: Using baseline data from a multicenter study in HIV-infected youth on stable treatment with cAR...

Transferrin receptor facilitates TGF-β and BMP signaling activation to control craniofacial morphogenesis

PubMed Central

Lei, R; Zhang, K; Liu, K; Shao, X; Ding, Z; Wang, F; Hong, Y; Zhu, M; Li, H; Li, H

2016-01-01

The Pierre Robin Sequence (PRS), consisting of cleft palate, glossoptosis and micrognathia, is a common human birth defect. However, how this abnormality occurs remains largely unknown. Here we report that neural crest cell (NCC)-specific knockout of transferrin receptor (Tfrc), a well known transferrin transporter protein, caused micrognathia, cleft palate, severe respiratory distress and inability to suckle in mice, which highly resemble human PRS. Histological and anatomical analysis revealed that the cleft palate is due to the failure of palatal shelves elevation that resulted from a retarded extension of Meckel's cartilage. Interestingly, Tfrc deletion dramatically suppressed both transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling in cranial NCCs-derived mandibular tissues, suggesting that Tfrc may act as a facilitator of these two signaling pathways during craniofacial morphogenesis. Together, our study uncovers an unknown function of Tfrc in craniofacial development and provides novel insight into the etiology of PRS. PMID:27362800

The Role of TIR-NBS and TIR-X Proteins in Plant Basal Defense Responses1[W][OA

PubMed Central

Nandety, Raja Sekhar; Caplan, Jeffery L.; Cavanaugh, Keri; Perroud, Bertrand; Wroblewski, Tadeusz; Michelmore, Richard W.; Meyers, Blake C.

2013-01-01

Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors. PMID:23735504

The role of TIR-NBS and TIR-X proteins in plant basal defense responses.

PubMed

Nandety, Raja Sekhar; Caplan, Jeffery L; Cavanaugh, Keri; Perroud, Bertrand; Wroblewski, Tadeusz; Michelmore, Richard W; Meyers, Blake C

2013-07-01

Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors.

Neurotrophic and Neurotoxic Effects of Amyloid |beta Protein: Reversal by Tachykinin Neuropeptides

NASA Astrophysics Data System (ADS)

Yankner, Bruce A.; Duffy, Lawrence K.; Kirschner, Daniel A.

1990-10-01

The amyloid β protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid β protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid β protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid β protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid β protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid β protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.

Identification of proteins likely to be involved in morphogenesis, cell division, and signal transduction in Planctomycetes by comparative genomics.

PubMed

Jogler, Christian; Waldmann, Jost; Huang, Xiaoluo; Jogler, Mareike; Glöckner, Frank Oliver; Mascher, Thorsten; Kolter, Roberto

2012-12-01

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a "guilt-by-association" approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.

Ocatin. A Novel Tuber Storage Protein from the Andean Tuber Crop Oca with Antibacterial and Antifungal Activities1

PubMed Central

Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E.

2002-01-01

The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens. PMID:11950978

Ocatin. A novel tuber storage protein from the andean tuber crop oca with antibacterial and antifungal activities.

PubMed

Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E

2002-04-01

The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens.

Fragile X Mental Retardation Syndrome: Structure of the KH1-KH2 Domains of Fragile X Mental Retardation Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valverde,R.; Poznyakova, I.; Kajander, T.

Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence ofmore » Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.« less

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity▿†

PubMed Central

Vanderlinde, Elizabeth M.; Magnus, Samantha A.; Tambalo, Dinah D.; Koval, Susan F.; Yost, Christopher K.

2011-01-01

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA+ ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella. PMID:21357485

Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from Arabidopsis thaliana in complex with AMP.

PubMed

Kim, Do Jin; Bitto, Eduard; Bingman, Craig A; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N

2015-07-01

Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/β overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Pyrophosphate-Dependent Fructose-6-Phosphate 1-Phosphotransferase Induction and Attenuation of Hsp Gene Expression during Endosperm Modification in Quality Protein Maize1[C][W][OA

PubMed Central

Guo, Xiaomei; Ronhovde, Kyla; Yuan, Lingling; Yao, Bo; Soundararajan, Madhavan P.; Elthon, Thomas; Zhang, Chi; Holding, David R.

2012-01-01

Quality Protein Maize (QPM) is a hard-endosperm version of the high-lysine opaque2 (o2) maize (Zea mays) mutant, but the genes involved in modification of the soft o2 endosperm are largely unknown. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the α-regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays showed a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to the wild type and o2. An expressed retrogene version of PFPα of unknown function that was not up-regulated in QPM was also identified. The elevated expression levels of a number of ATP-requiring heat shock proteins (Hsps) in o2 endosperm are ameliorated in QPM. PFPα is also coinduced with Hsps in maize roots in response to heat, cold, and the unfolded protein response stresses. We propose that reduced ATP availability resulting from the generalized Hsp response in addition to the reduction of pyruvate, orthophosphate dikinase activity in o2 endosperm is compensated in part by increased PFP activity in QPM. PMID:22158678

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bossi, Flavia; Fan, Jue; Xiao, Jun

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE PAGES

Bossi, Flavia; Fan, Jue; Xiao, Jun; ...

2017-06-26

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

Molecular Convergence of Infrared Vision in Snakes

PubMed Central

Yokoyama, Shozo; Altun, Ahmet; DeNardo, Dale F.

2011-01-01

It has been discovered that the transient receptor potential ankyrin 1 (TRPA1) proteins of Boidae (boas), Pythonidae (pythons), and Crotalinae (pit vipers) are used to detect infrared radiation, but the molecular mechanism for detecting the infrared radiation is unknown. Here, relating the amino acid substitutions in their TRPA1 proteins and the functional differentiations, we propose that three parallel amino acid changes (L330M, Q391H, and S434T) are responsible for the development of infrared vision in the three groups of snakes. Protein modeling shows that the three amino acid changes alter the structures of the central region of their ankyrin repeats. PMID:20937734

Widespread genetic switches and toxicity resistance proteins for fluoride.

PubMed

Baker, Jenny L; Sudarsan, Narasimhan; Weinberg, Zasha; Roth, Adam; Stockbridge, Randy B; Breaker, Ronald R

2012-01-13

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

PubMed Central

Weinberg, Zasha; Roth, Adam; Stockbridge, Randy B.; Breaker, Ronald R.

2014-01-01

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion. PMID:22194412

Interspecific and host-related gene expression patterns in nematode-trapping fungi.

PubMed

Andersson, Karl-Magnus; Kumar, Dharmendra; Bentzer, Johan; Friman, Eva; Ahrén, Dag; Tunlid, Anders

2014-11-11

Nematode-trapping fungi are soil-living fungi that capture and kill nematodes using special hyphal structures called traps. They display a large diversity of trapping mechanisms and differ in their host preferences. To provide insights into the genetic basis for this variation, we compared the transcriptome expressed by three species of nematode-trapping fungi (Arthrobotrys oligospora, Monacrosporium cionopagum and Arthrobotrys dactyloides, which use adhesive nets, adhesive branches or constricting rings, respectively, to trap nematodes) during infection of two different plant-pathogenic nematode hosts (the root knot nematode Meloidogyne hapla and the sugar beet cyst nematode Heterodera schachtii). The divergence in gene expression between the fungi was significantly larger than that related to the nematode species being infected. Transcripts predicted to encode secreted proteins and proteins with unknown function (orphans) were overrepresented among the highly expressed transcripts in all fungi. Genes that were highly expressed in all fungi encoded endopeptidases, such as subtilisins and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors; and transcripts containing the Ricin-B lectin domain. Differentially expressed transcripts among the fungal species encoded various lectins, such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain; and proteins containing a DUF3129 domain. A small set of transcripts were differentially expressed in infections of different host nematodes, including peptidases, WSC domain proteins, tyrosinases, and small secreted proteins with unknown function. This is the first study on the variation of infection-related gene expression patterns in nematode-trapping fungi infecting different host species. A better understanding of these patterns will facilitate the improvements of these fungi in biological control programs, by providing molecular markers for screening programs and candidates for genetic manipulations of virulence and host preferences.

Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function

PubMed Central

Grobei, Monica A.; Qeli, Ermir; Brunner, Erich; Rehrauer, Hubert; Zhang, Runxuan; Roschitzki, Bernd; Basler, Konrad; Ahrens, Christian H.; Grossniklaus, Ueli

2009-01-01

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues. PMID:19546170

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation*

PubMed Central

Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W.; Wanders, Ronald J. A.; Locasale, Jason W.; Lombard, David B.; de Boer, Vincent C. J.; Zhao, Yingming

2015-01-01

The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD−/− fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

PubMed Central

Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Céline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J.M.; Pla, Alessandra Fiorio; Prevarskaya, Natalia

2015-01-01

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186

Characterization of the Mouse Pancreatic Islet Proteome and Comparative Analysis with Other Mouse Tissues

PubMed Central

Petyuk, Vladislav A.; Qian, Wei-Jun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

2009-01-01

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of type 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2,612 proteins having at least two unique peptides per protein. The dataset also identified ~60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at http://ncrr.pnl.gov, provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields. PMID:18570455

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae).

PubMed

Uhlig, Christiane; Kabisch, Johannes; Palm, Gottfried J; Valentin, Klaus; Schweder, Thomas; Krell, Andreas

2011-12-01

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability. Copyright © 2011 Elsevier Inc. All rights reserved.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

PubMed

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

PubMed Central

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

Probing the functions of long non-coding RNAs by exploiting the topology of global association and interaction network.

PubMed

Deng, Lei; Wu, Hongjie; Liu, Chuyao; Zhan, Weihua; Zhang, Jingpu

2018-06-01

Long non-coding RNAs (lncRNAs) are involved in many biological processes, such as immune response, development, differentiation and gene imprinting and are associated with diseases and cancers. But the functions of the vast majority of lncRNAs are still unknown. Predicting the biological functions of lncRNAs is one of the key challenges in the post-genomic era. In our work, We first build a global network including a lncRNA similarity network, a lncRNA-protein association network and a protein-protein interaction network according to the expressions and interactions, then extract the topological feature vectors of the global network. Using these features, we present an SVM-based machine learning approach, PLNRGO, to annotate human lncRNAs. In PLNRGO, we construct a training data set according to the proteins with GO annotations and train a binary classifier for each GO term. We assess the performance of PLNRGO on our manually annotated lncRNA benchmark and a protein-coding gene benchmark with known functional annotations. As a result, the performance of our method is significantly better than that of other state-of-the-art methods in terms of maximum F-measure and coverage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells.

PubMed

Lyupina, Yulia V; Erokhov, Pavel A; Kravchuk, Oksana I; Finoshin, Alexander D; Abaturova, Svetlana B; Orlova, Olga V; Beljelarskaya, Svetlana N; Kostyuchenko, Margarita V; Mikhailov, Victor S

2018-06-08

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system. Copyright © 2018 Elsevier B.V. All rights reserved.

BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis.

PubMed

Peng, Yuancheng; Chen, Liangliang; Li, Shengjun; Zhang, Yueying; Xu, Ran; Liu, Zupei; Liu, Wuxia; Kong, Jingjing; Huang, Xiahe; Wang, Yingchun; Cheng, Beijiu; Zheng, Leiying; Li, Yunhai

2018-04-18

Sugars function as signal molecules to regulate growth, development, and gene expression in plants, yeasts, and animals. A coordination of sugar availability with phytohormone signals is crucial for plant growth and development. The molecular link between sugar availability and hormone-dependent plant growth are largely unknown. Here we report that BRI1 and BAK1 are involved in sugar-responsive growth and development. Glucose influences the physical interactions and phosphorylations of BRI1 and BAK1 in a concentration-dependent manner. BRI1 and BAK1 physically interact with G proteins that are essential for mediating sugar signaling. Biochemical data show that BRI1 can phosphorylate G protein β subunit and γ subunits, and BAK1 can phosphorylate G protein γ subunits. Genetic analyses suggest that BRI1 and BAK1 function in a common pathway with G-protein subunits to regulate sugar responses. Thus, our findings reveal an important genetic and molecular mechanism by which BR receptors associate with G proteins to regulate sugar-responsive growth and development.

Changes in hippocampal synaptic functions and protein expression in monosodium glutamate-treated obese mice during development of glucose intolerance.

PubMed

Sasaki-Hamada, Sachie; Hojo, Yuki; Koyama, Hajime; Otsuka, Hayuma; Oka, Jun-Ichiro

2015-05-01

Glucose is the sole neural fuel for the brain and is essential for cognitive function. Abnormalities in glucose tolerance may be associated with impairments in cognitive function. Experimental obese model mice can be generated by an intraperitoneal injection of monosodium glutamate (MSG; 2 mg/g) once a day for 5 days from 1 day after birth. MSG-treated mice have been shown to develop glucose intolerance and exhibit chronic neuroendocrine dysfunction associated with marked cognitive malfunctions at 28-29  weeks old. Although hippocampal synaptic plasticity is impaired in MSG-treated mice, changes in synaptic transmission remain unknown. Here, we investigated whether glucose intolerance influenced cognitive function, synaptic properties and protein expression in the hippocampus. We demonstrated that MSG-treated mice developed glucose intolerance due to an impairment in the effectiveness of insulin actions, and showed cognitive impairments in the Y-maze test. Moreover, long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal synapses in hippocampal slices was impaired, and the relationship between the slope of extracellular field excitatory postsynaptic potential and stimulus intensity of synaptic transmission was weaker in MSG-treated mice. The protein levels of vesicular glutamate transporter 1 and GluA1 glutamate receptor subunits decreased in the CA1 region of MSG-treated mice. These results suggest that deficits in glutamatergic presynapses as well as postsynapses lead to impaired synaptic plasticity in MSG-treated mice during the development of glucose intolerance, though it remains unknown whether impaired LTP is due to altered inhibitory transmission. It may be important to examine changes in glucose tolerance in order to prevent cognitive malfunctions associated with diabetes. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

PubMed Central

Mahdessian, Hovsep; Taxiarchis, Apostolos; Popov, Sergej; Silveira, Angela; Franco-Cereceda, Anders; Hamsten, Anders; Eriksson, Per; van't Hooft, Ferdinand

2014-01-01

Genome-wide association studies have identified a locus on chromosome 19 associated with plasma triglyceride (TG) concentration and nonalcoholic fatty liver disease. However, the identity and functional role of the gene(s) responsible for these associations remain unknown. Of 19 expressed genes contained in this locus, none has previously been implicated in lipid metabolism. We performed gene expression studies and expression quantitative trait locus analysis in 206 human liver samples to identify the putative causal gene. Transmembrane 6 superfamily member 2 (TM6SF2), a gene with hitherto unknown function, expressed predominantly in liver and intestine, was identified as the putative causal gene. TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane domains. Otherwise, no other protein features were identified which could help to elucidate the function of TM6SF2. Protein subcellular localization studies with confocal microscopy demonstrated that TM6SF2 is localized in the endoplasmic reticulum and the ER-Golgi intermediate compartment of human liver cells. Functional studies for secretion of TG-rich lipoproteins (TRLs) and lipid droplet content were performed in human hepatoma Huh7 and HepG2 cells using confocal microscopy and siRNA inhibition and overexpression techniques. In agreement with the genome-wide association data, it was found that TM6SF2 siRNA inhibition was associated with reduced secretion of TRLs and increased cellular TG concentration and lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis. We conclude that TM6SF2 is a regulator of liver fat metabolism with opposing effects on the secretion of TRLs and hepatic lipid droplet content. PMID:24927523

Management of Gene Variants of Unknown Significance: Analysis Method and Risk Assessment of the VHL Mutation p.P81S (c.241C>T).

PubMed

Alosi, Daniela; Bisgaard, Marie Luise; Hemmingsen, Sophie Nowak; Krogh, Lotte Nylandsted; Mikkelsen, Hanne Birte; Binderup, Marie Louise Mølgaard

2017-02-01

Evaluation of the pathogenicity of a gene variant of unknown significance (VUS) is crucial for molecular diagnosis and genetic counseling, but can be challenging. This is especially so in phenotypically variable diseases, such as von Hippel-Lindau disease (vHL). vHL is caused by germline mutations in the VHL gene, which predispose to the development of multiple tumors such as central nervous system hemangioblastomas and renal cell carcinoma (RCC). We propose a method for the evaluation of VUS pathogenicity through our experience with the VHL missense mutation c.241C>T (p.P81S). 1) Clinical evaluation of known variant carriers: We evaluated a family of five VHL p.P81S carriers, as well as the clinical characteristics of all the p.P81S carriers reported in the literature; 2) Evaluation of tumor tissue via genetic analysis, histology, and immunohistochemistry (IHC); 3) Assessment of the variant's impact on protein structure and function, using multiple databases, in silico algorithms, and reports of functional studies. Only one family member had clinical signs of vHL with early-onset RCC. IHC analysis showed no VHL protein expressed in the tumor, consistent with biallelic VHL inactivation. The majority of in silico algorithms reported p.P81S as possibly pathogenic in relation to vHL or RCC, but there were discrepancies. Functional studies suggest that p.P81S impairs the VHL protein's function. The VHL p.P81S mutation is most likely a low-penetrant pathogenic variant predisposing to RCC development. We suggest the above-mentioned method for VUS evaluation with use of different methods, especially a variety of in silico methods and tumor tissue analysis.

Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.

PubMed

Brockly, Frédérique; Piechaczyk, Marc; Bossis, Guillaume

2016-01-01

SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.

Retention mechanisms for ER and Golgi membrane proteins.

PubMed

Gao, Caiji; Cai, Yi; Wang, Yejun; Kang, Byung-Ho; Aniento, Fernando; Robinson, David G; Jiang, Liwen

2014-08-01

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2.

PubMed

Van Huffel, S; Delaei, F; Heyninck, K; De Valck, D; Beyaert, R

2001-08-10

The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.

A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

PubMed

Vigil, Dominico; Lin, Jung-Hsin; Sotriffer, Christoph A; Pennypacker, Juniper K; McCammon, J Andrew; Taylor, Susan S

2006-01-01

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

PubMed

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

PubMed Central

O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.

2009-01-01

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564

TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding

PubMed Central

Sakaguchi, Masakiyo; Murata, Hitoshi; Yamamoto, Ken-ichi; Ono, Tomoyuki; Sakaguchi, Yoshihiko; Motoyama, Akira; Hibino, Toshihiko; Kataoka, Ken; Huh, Nam-ho

2011-01-01

The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions. PMID:21829704

Neuroglobin and cytoglobin: two new members of globin family

PubMed Central

Tosqui, Priscilla; Colombo, Marcio Francisco

2011-01-01

The globin family has long been defined by myoglobin and hemoglobin, proteins with the functions of oxygen storage and transportation, respectively. Recently, two new members of this family were discovered: neuroglobin present in neurons and retinal cells and cytoglobin found in various types of tissue. The increased expression of these proteins in hypoxic conditions first suggested a role in oxygen supply. However structural and functional differences, such as the hexacoordinated heme, a high autoxidation rate and different concentrations between different cellular types, have dismissed this hypothesis. The protective role of these globins has already been established. In vitro and in vivo studies have demonstrated increased survival of neurons under stress in the presence of neuroglobin and increased resistance to neurodegenerative diseases. However the mechanism remains unknown. Functions, including detoxification of nitric oxide, free radical scavenging and as an antioxidant and signaling of apoptosis, have also been suggested for neuroglobin and an antifibrotic function for cytoglobin. PMID:23049323

Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

PubMed

Dhar, Jayeeta; Barik, Sailen

2016-12-01

Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Roslyn N.; Sanford, James A.; Park, Jea H.

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators ofmore » two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.« less

A novel astaxanthin-binding photooxidative stress-inducible aqueous carotenoprotein from a eukaryotic microalga isolated from asphalt in midsummer.

PubMed

Kawasaki, Shinji; Mizuguchi, Keisuke; Sato, Masaru; Kono, Tetsuya; Shimizu, Hirofumi

2013-07-01

Water-soluble orange carotenoid proteins (OCPs) that bind 3'-hydroxyechinenone are found in cyanobacteria, and are thought to play a key role in photoprotection. The distribution of OCPs in eukaryotes remains largely unknown. In this study, we identified a novel OCP that predominantly binds astaxanthin from a eukaryotic microalga, strain Ki-4, isolated from a dry surface of heated asphalt in midsummer. A purified astaxanthin-binding OCP, named AstaP, shows high solubility in water with an absorption peak at 484 nm, and possesses a heat-stable activity that quenches singlet oxygen. The deduced amino acid sequence of AstaP comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of unknown function are distributed mainly in organisms from the hydrosphere, such as marine bacteria, cyanobacteria, sea anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI) value among homologs. The gene encoding AstaP, as well as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high light exposure. As a unique aqueous carotenoprotein, AstaP will provide a novel function of OCPs in protection against extreme photooxidative stresses.

Persistence of Functional Protein Domains in Mycoplasma Species and their Role in Host Specificity and Synthetic Minimal Life.

PubMed

Kamminga, Tjerko; Koehorst, Jasper J; Vermeij, Paul; Slagman, Simen-Jan; Martins Dos Santos, Vitor A P; Bijlsma, Jetta J E; Schaap, Peter J

2017-01-01

Mycoplasmas are the smallest self-replicating organisms and obligate parasites of a specific vertebrate host. An in-depth analysis of the functional capabilities of mycoplasma species is fundamental to understand how some of simplest forms of life on Earth succeeded in subverting complex hosts with highly sophisticated immune systems. In this study we present a genome-scale comparison, focused on identification of functional protein domains, of 80 publically available mycoplasma genomes which were consistently re-annotated using a standardized annotation pipeline embedded in a semantic framework to keep track of the data provenance. We examined the pan- and core-domainome and studied predicted functional capability in relation to host specificity and phylogenetic distance. We show that the pan- and core-domainome of mycoplasma species is closed. A comparison with the proteome of the "minimal" synthetic bacterium JCVI-Syn3.0 allowed us to classify domains and proteins essential for minimal life. Many of those essential protein domains, essential Domains of Unknown Function (DUFs) and essential hypothetical proteins are not persistent across mycoplasma genomes suggesting that mycoplasma species support alternative domain configurations that bypass their essentiality. Based on the protein domain composition, we could separate mycoplasma species infecting blood and tissue. For selected genomes of tissue infecting mycoplasmas, we could also predict whether the host is ruminant, pig or human. Functionally closely related mycoplasma species, which have a highly similar protein domain repertoire, but different hosts could not be separated. This study provides a concise overview of the functional capabilities of mycoplasma species, which can be used as a basis to further understand host-pathogen interaction or to design synthetic minimal life.

Persistence of Functional Protein Domains in Mycoplasma Species and their Role in Host Specificity and Synthetic Minimal Life

PubMed Central

Kamminga, Tjerko; Koehorst, Jasper J.; Vermeij, Paul; Slagman, Simen-Jan; Martins dos Santos, Vitor A. P.; Bijlsma, Jetta J. E.; Schaap, Peter J.

2017-01-01

Mycoplasmas are the smallest self-replicating organisms and obligate parasites of a specific vertebrate host. An in-depth analysis of the functional capabilities of mycoplasma species is fundamental to understand how some of simplest forms of life on Earth succeeded in subverting complex hosts with highly sophisticated immune systems. In this study we present a genome-scale comparison, focused on identification of functional protein domains, of 80 publically available mycoplasma genomes which were consistently re-annotated using a standardized annotation pipeline embedded in a semantic framework to keep track of the data provenance. We examined the pan- and core-domainome and studied predicted functional capability in relation to host specificity and phylogenetic distance. We show that the pan- and core-domainome of mycoplasma species is closed. A comparison with the proteome of the “minimal” synthetic bacterium JCVI-Syn3.0 allowed us to classify domains and proteins essential for minimal life. Many of those essential protein domains, essential Domains of Unknown Function (DUFs) and essential hypothetical proteins are not persistent across mycoplasma genomes suggesting that mycoplasma species support alternative domain configurations that bypass their essentiality. Based on the protein domain composition, we could separate mycoplasma species infecting blood and tissue. For selected genomes of tissue infecting mycoplasmas, we could also predict whether the host is ruminant, pig or human. Functionally closely related mycoplasma species, which have a highly similar protein domain repertoire, but different hosts could not be separated. This study provides a concise overview of the functional capabilities of mycoplasma species, which can be used as a basis to further understand host-pathogen interaction or to design synthetic minimal life. PMID:28224116

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

PubMed Central

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-01-01

Background Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. Results As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. Conclusion We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s). PMID:19055778

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis.

PubMed

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-12-03

Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, approximately 4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).

Comparison of the protein-coding gene content of Chlamydia trachomatis and Protochlamydia amoebophila using a Raspberry Pi computer.

PubMed

Robson, James F; Barker, Daniel

2015-10-13

To demonstrate the bioinformatics capabilities of a low-cost computer, the Raspberry Pi, we present a comparison of the protein-coding gene content of two species in phylum Chlamydiae: Chlamydia trachomatis, a common sexually transmitted infection of humans, and Candidatus Protochlamydia amoebophila, a recently discovered amoebal endosymbiont. Identifying species-specific proteins and differences in protein families could provide insights into the unique phenotypes of the two species. Using a Raspberry Pi computer, sequence similarity-based protein families were predicted across the two species, C. trachomatis and P. amoebophila, and their members counted. Examples include nine multi-protein families unique to C. trachomatis, 132 multi-protein families unique to P. amoebophila and one family with multiple copies in both. Most families unique to C. trachomatis were polymorphic outer-membrane proteins. Additionally, multiple protein families lacking functional annotation were found. Predicted functional interactions suggest one of these families is involved with the exodeoxyribonuclease V complex. The Raspberry Pi computer is adequate for a comparative genomics project of this scope. The protein families unique to P. amoebophila may provide a basis for investigating the host-endosymbiont interaction. However, additional species should be included; and further laboratory research is required to identify the functions of unknown or putative proteins. Multiple outer membrane proteins were found in C. trachomatis, suggesting importance for host evasion. The tyrosine transport protein family is shared between both species, with four proteins in C. trachomatis and two in P. amoebophila. Shared protein families could provide a starting point for discovery of wide-spectrum drugs against Chlamydiae.

Combined copper/zinc attachment to prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Bernholc, Jerry

2013-03-01

Misfolding of prion protein (PrP) is responsible for diseases such as ``mad-cow disease'' in cattle and Creutzfeldt-Jacob in humans. Extensive experimental investigation has established that this protein strongly interacts with copper ions, and this ability has been linked to its still unknown function. Attachment of other metal ions (zinc, iron, manganese) have been demonstrated as well, but none of them could outcompete copper. Recent finding, however, indicates that at intermediate concentrations both copper and zinc ions can attach to the PrP at the octarepeat region, which contains high affinity metal binding sites. Based on this evidence, we have performed density functional theory simulations to investigate the combined Cu/Zn attachment. We consider all previously reported binding modes of copper at the octarepeat region and examine a possibility simultaneous Cu/Zn attachment. We find that this can indeed occur for only one of the known binding sites, when copper changes its coordination mode to allow for attachment of zinc ion. The implications of the simultaneous attachment on neural function remain to be explored.

Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.

PubMed

Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina; Chagas da Cunha, Julia Pinheiro

2017-01-01

Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

PubMed

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Svedas, Vytas

2014-07-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure-function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 71201[OPEN

PubMed Central

López-Igual, Rocío; Wilson, Adjélé; Bourcier de Carbon, Céline; Sutter, Markus; Turmo, Aiko

2016-01-01

The photoactive Orange Carotenoid Protein (OCP) is involved in cyanobacterial photoprotection. Its N-terminal domain (NTD) is responsible for interaction with the antenna and induction of excitation energy quenching, while the C-terminal domain is the regulatory domain that senses light and induces photoactivation. In most nitrogen-fixing cyanobacterial strains, there are one to four paralogous genes coding for homologs to the NTD of the OCP. The functions of these proteins are unknown. Here, we study the expression, localization, and function of these genes in Anabaena sp. PCC 7120. We show that the four genes present in the genome are expressed in both vegetative cells and heterocysts but do not seem to have an essential role in heterocyst formation. This study establishes that all four Anabaena NTD-like proteins can bind a carotenoid and the different paralogs have distinct functions. Surprisingly, only one paralog (All4941) was able to interact with the antenna and to induce permanent thermal energy dissipation. Two of the other Anabaena paralogs (All3221 and Alr4783) were shown to be very good singlet oxygen quenchers. The fourth paralog (All1123) does not seem to be involved in photoprotection. Structural homology modeling allowed us to propose specific features responsible for the different functions of these soluble carotenoid-binding proteins. PMID:27208286

Identification of functional interactome of a key cell division regulatory protein CedA of E.coli.

PubMed

Sharma, Pankaj; Tomar, Anil Kumar; Kundu, Bishwajit

2018-01-01

Cell division is compromised in DnaAcos mutant Escherichia coli cells that results in filamentous cell morphology. This is countered by over-expression of CedA protein that induces cytokinesis and thus, regular cell morphology is regained; however via an unknown mechanism. To understand the process systematically, exact role of CedA should be deciphered. Protein interactions are crucial for functional organization of a cell and their identification helps in revealing exact function(s) of a protein and its binding partners. Thus, this study was intended to identify CedA binding proteins (CBPs) to gain more clues of CedA function. We isolated CBPs by pull down assay using purified recombinant CedA and identified nine CBPs by mass spectrometric analysis (MALDI-TOF MS and LC-MS/MS), viz. PDHA1, RL2, DNAK, LPP, RPOB, G6PD, GLMS, RL3 and YBCJ. Based on CBPs identified, we hypothesize that CedA plays a crucial and multifaceted role in cell cycle regulation and specific pathways in which CedA participates may include transcription and energy metabolism. However, further validation through in-vitro and in-vivo experiments is necessary. In conclusion, identification of CBPs may help us in deciphering mechanism of CedA mediated cell division during chromosomal DNA over-replication. Copyright © 2017 Elsevier B.V. All rights reserved.

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

PubMed Central

Sitaram, Anand; Piccirillo, Rosanna; Palmisano, Ilaria; Harper, Dawn C.; Dell'Angelica, Esteban C.; Schiaffino, M. Vittoria

2009-01-01

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes. PMID:19116314

Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis*

PubMed Central

Shchedrina, Valentina A.; Everley, Robert A.; Zhang, Yan; Gygi, Steven P.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Selenoprotein K (SelK) is an 11-kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, selenoprotein S (SelS), and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins, and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had higher affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element, and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, and STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the oligosaccharyltransferase complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes. PMID:22016385

Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily.

PubMed

Lakshmi, Balasubramanian; Mishra, Madhulika; Srinivasan, Narayanaswamy; Archunan, Govindaraju

2015-01-01

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

PubMed Central

Roche, John P.; Alsharif, Peter; Graf, Ethan R.

2015-01-01

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function. PMID:26317909

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

PubMed Central

Näf, Dieter; Kupfer, Gary M.; Suliman, Ahmed; Lambert, Kathleen; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability. PMID:9742112

ISG15 in the tumorigenesis and treatment of cancer: An emerging role in malignancies of the digestive system

PubMed Central

Zuo, Chaohui; Sheng, Xinyi; Ma, Min; Xia, Man; Ouyang, Linda

2016-01-01

The interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) encodes an IFN-inducible, ubiquitin-like protein. The ISG15 protein forms conjugates with numerous cellular proteins that are involved in a multitude of cellular functions, including interferon-induced immune responses and the regulation of cellular protein turnover. The expression of ISG15 and ISG15-mediated conjugation has been implicated in a wide range of human tumors and cancer cell lines, but the roles of ISG15 in tumorigenesis and responses to anticancer treatments remain largely unknown. In this review, we discuss the findings of recent studies with regard to the role of ISG15 pathways in cancers of the digestive system. PMID:27626310

1H, 13C, and 15N resonance assignments for the protein coded by gene locus BB0938 of Bordetella bronchiseptica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rossi, Paolo; Ramelot, Theresa A.; Xiao, Rong

2005-11-01

The product of gene locus BB0938 from Bordetella bronchiseptica (Swiss-Prot ID: Q7WNU7-BORBR; NESG target ID: BoR11; Wunderlich et al., 2004; Pfam ID: PF03476) is a 128-residue protein of unknown function. This broadly conserved protein family is found in eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 98% of backbone and 94% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a b topology with a seven-residue helical insert, ??????????. BMRB deposit with accession number 6693. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

1H, 13C, and 15N resonance assignments for Escherichia coli ytfP, a member of the broadly conserved UPF0131 protein domain family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aramini, James M.; Swapna, G.V.T.; Huang, Yuanpeng

2005-11-01

Protein ytfP from Escherichia coli (Swiss-Prot ID: YTFP-ECOLI; NESG target ID: ER111; Wunderlich et al., 2004) is a 113-residue member of the UPF0131 protein family (Pfam ID: PF03674) of unknown function. This domain family is found in organisms from all three kingdoms, archaea, eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 97% of backbone and 91% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a mixed a/b topology,????????. BMRB deposit with Accession No. 6448. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

ISG15 in the tumorigenesis and treatment of cancer: An emerging role in malignancies of the digestive system.

PubMed

Zuo, Chaohui; Sheng, Xinyi; Ma, Min; Xia, Man; Ouyang, Linda

2016-11-08

The interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) encodes an IFN-inducible, ubiquitin-like protein. The ISG15 protein forms conjugates with numerous cellular proteins that are involved in a multitude of cellular functions, including interferon-induced immune responses and the regulation of cellular protein turnover. The expression of ISG15 and ISG15-mediated conjugation has been implicated in a wide range of human tumors and cancer cell lines, but the roles of ISG15 in tumorigenesis and responses to anticancer treatments remain largely unknown. In this review, we discuss the findings of recent studies with regard to the role of ISG15 pathways in cancers of the digestive system.

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta.

PubMed

Nausch, I; Mentlein, R; Heymann, E

1990-11-01

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa

The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals.more » It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric 'lids' that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins.« less

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

PubMed

Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

2003-10-01

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Precursor-product discrimination by La protein during tRNA metabolism

PubMed Central

Bayfield, Mark A.; Maraia, Richard J.

2009-01-01

SUMMARY La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. While the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA-binding β-sheet surface of RRM1 is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 β surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding while processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA but not UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair a RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA. PMID:19287396

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

Membrane and inclusion body targeting of lyssavirus matrix proteins.

PubMed

Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

2013-02-01

Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

Replacement of the respiratory syncytial virus nonstructural proteins NS1 and NS2 by the V protein of parainfluenza virus 5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tran, Kim C.; He, Biao; Teng, Michael N.

2007-11-10

Paramyxoviruses have been shown to produce proteins that inhibit interferon production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to have interferon antagonist activity through an unknown mechanism. To understand further the functions of NS1 and NS2, we generated recombinant RSV in which both NS1 and NS2 were replaced by the PIV5 V protein, which has well-characterized IFN antagonist activities ({delta}NS1/2-V). Expression of V was able to partially inhibit IFN responses in {delta}NS1/2-V-infected cells. In addition, the replication kinetics of {delta}NS1/2-V were intermediate between {delta}NS1/2 and wild-type (rA2) in A549 cells.more » However, expression of V did not affect the ability of {delta}NS1/2-V to activate IRF3 nuclear translocation and IFN{beta} transcription. These data indicate that V was able to replace some of the IFN inhibitory functions of the RSV NS1 and NS2 proteins, but also that NS1 and NS2 have functions in viral replication beyond IFN antagonism.« less

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

PubMed

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

PubMed

Recouvreux, Pierre; Sokolowski, Thomas R; Grammoustianou, Aristea; ten Wolde, Pieter Rein; Dogterom, Marileen

2016-02-16

Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns.

Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein-Protein Interaction Data.

PubMed

Pokharel, Yuba Raj; Saarela, Jani; Szwajda, Agnieszka; Rupp, Christian; Rokka, Anne; Lal Kumar Karna, Shibendra; Teittinen, Kaisa; Corthals, Garry; Kallioniemi, Olli; Wennerberg, Krister; Aittokallio, Tero; Westermarck, Jukka

2015-12-01

High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges in translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a relevance ranking platform (RRP), which consist of modular functional and bioinformatic filters to provide relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data, highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validated the importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition. The RRP framework is modular and can be modified to answer versatile research problems depending on the nature of the biological question under study. Based on comparison of RRP to other existing filtering tools, the presented data indicate that RRP offers added value especially for the analysis of interacting proteins for which there is no sufficient prior knowledge available. Finally, we encourage the use of RRP in combination with either SAINT or CRAPome computational tools for selecting the candidate interactors that fulfill the both important requirements, functional relevance, and high confidence interaction detection. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2015-08-01

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

A core viral protein binds host nucleosomes to sequester immune danger signals

PubMed Central

Avgousti, Daphne C.; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J.; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C.; Blumenthal, Daniel; Paris, Andrew J.; Reyes, Emigdio D.; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H.; Worthen, G. Scott; Black, Ben E.; Garcia, Benjamin A.; Weitzman, Matthew D.

2016-01-01

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling. PMID:27362237

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling

PubMed Central

Zhou, Jian-Ying; Krovvidi, Ravi K.; Gao, Yuqian; Gao, Hong; Petritis, Brianne O.; De, Asit; Miller-Graziano, Carol; Bankey, Paul E.; Petyuk, Vladislav A.; Nicora, Carrie D.; Clauss, Therese R; Moore, Ronald J.; Shi, Tujin; Brown, Joseph N.; Kaushal, Amit; Xiao, Wenzhong; Davis, Ronald W.; Maier, Ronald V.; Tompkins, Ronald G.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.

2013-01-01

PURPOSE Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. EXPERIMENTAL DESIGN We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. RESULTS A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. CONCLUSIONS AND CLINICAL RELEVANCE Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs. PMID:23589343

The three-dimensional structure of "Lonely Guy" from Claviceps purpurea provides insights into the phosphoribohydrolase function of Rossmann fold-containing lysine decarboxylase-like proteins.

PubMed

Dzurová, Lenka; Forneris, Federico; Savino, Simone; Galuszka, Petr; Vrabka, Josef; Frébort, Ivo

2015-08-01

The recently discovered cytokinin (CK)-specific phosphoribohydrolase "Lonely Guy" (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function. © 2015 Wiley Periodicals, Inc.

The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

PubMed Central

Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J

2017-01-01

Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with AMPARs in artificial expression systems, but it is unknown whether Shisa7 has a functional role in glutamatergic synapses. We show that Shisa7 physically interacts with synaptic AMPARs in mouse hippocampus. Shisa7 gene deletion resulted in faster AMPAR currents in CA1 synapses, without affecting its synaptic expression. Shisa7 KO mice showed reduced initiation and maintenance of long-term potentiation of glutamatergic synapses. In line with this, Shisa7 KO mice showed a specific deficit in contextual fear memory, both short-term and long-term after conditioning, whereas auditory fear memory and anxiety-related behavior were normal. Thus, Shisa7 is a bona-fide AMPAR modulatory protein affecting channel kinetics of AMPARs, necessary for synaptic hippocampal plasticity, and memory recall. PMID:29199957

Complete fold annotation of the human proteome using a novel structural feature space

DOE PAGES

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

2017-04-13

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this methodmore » by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Finally, our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.« less

Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions

DOE PAGES

Jiang, Jiansen; Chan, Henry; Cash, Darian D.; ...

2015-10-15

Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). In this paper, we report the cryo–electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunitmore » interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Finally, our findings provide structural and mechanistic insights into telomerase holoenzyme function.« less

Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jiansen; Chan, Henry; Cash, Darian D.

Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). In this paper, we report the cryo–electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunitmore » interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Finally, our findings provide structural and mechanistic insights into telomerase holoenzyme function.« less

The interaction between NOLC1 and IAV NS1 protein promotes host cell apoptosis and reduces virus replication.

PubMed

Zhu, Chunyu; Zheng, Fangliang; Zhu, Junfeng; Liu, Meichen; Liu, Na; Li, Xue; Zhang, Li; Deng, Zaidong; Zhao, Qi; Liu, Hongsheng

2017-11-07

NS1 of the influenza virus plays an important role in the infection ability of the influenza virus. Our previous research found that NS1 protein interacts with the NOLC1 protein of host cells, however, the function of the interaction is unknown. In the present study, the role of the interaction between the two proteins in infection was further studied. Several analyses, including the use of a pull-down assay, Co-IP, western blot analysis, overexpression, RNAi, flow cytometry, etc., were used to demonstrate that the NS1 protein of H3N2 influenza virus interacts with host protein NOLC1 and reduces the quantity of NOLC1. The interaction also promotes apoptosis in A549 host cells, while the suppression of NOLC1 protein reduces the proliferation of the H3N2 virus. Based on these data, it was concluded that during the process of infection, NS1 protein interacts with NOLC1 protein, reducing the level of NOLC1, and that the interaction between the two proteins promotes apoptosis of host cells, thus reducing the proliferation of the virus. These findings provide new information on the biological function of the interaction between NS1 and NOLC1.

Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

PubMed

Zhang, Bin; Qiu, Han-Lin; Qu, Dong-Hai; Ruan, Ying; Chen, Dong-Hong

2018-04-05

Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wanitchang, Asawin; Wongthida, Phonphimon; Jongkae

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fusedmore » with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.« less

Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

PubMed Central

Qin, Xiao-Song; Shono, Akemi; Yamamoto, Akitsugu; Kurihara, Hidetake; Doi, Toshio

2009-01-01

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury. PMID:19850954

Arabidopsis  SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity.

PubMed

Pietra, Stefano; Gustavsson, Anna; Kiefer, Christian; Kalmbach, Lothar; Hörstedt, Per; Ikeda, Yoshihisa; Stepanova, Anna N; Alonso, Jose M; Grebe, Markus

2013-01-01

The orientation of cell division and the coordination of cell polarity within the plane of the tissue layer (planar polarity) contribute to shape diverse multicellular organisms. The root of Arabidopsis thaliana displays regularly oriented cell divisions, cell elongation and planar polarity providing a plant model system to study these processes. Here we report that the SABRE protein, which shares similarity with proteins of unknown function throughout eukaryotes, has important roles in orienting cell division and planar polarity. SABRE localizes at the plasma membrane, endomembranes, mitotic spindle and cell plate. SABRE stabilizes the orientation of CLASP-labelled preprophase band microtubules predicting the cell division plane, and of cortical microtubules driving cell elongation. During planar polarity establishment, sabre is epistatic to clasp at directing polar membrane domains of Rho-of-plant GTPases. Our findings mechanistically link SABRE to CLASP-dependent microtubule organization, shedding new light on the function of SABRE-related proteins in eukaryotes.

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function

PubMed Central

Beigi, Farideh; Gonzalez, Daniel R.; Minhas, Khalid M.; Sun, Qi-An; Foster, Matthew W.; Khan, Shakil A.; Treuer, Adriana V.; Dulce, Raul A.; Harrison, Robert W.; Saraiva, Roberto M.; Premer, Courtney; Schulman, Ivonne Hernandez; Stamler, Jonathan S.; Hare, Joshua M.

2012-01-01

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist–induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation. PMID:22366318

Sam37 is crucial for formation of the mitochondrial TOM-SAM supercomplex, thereby promoting β-barrel biogenesis.

PubMed

Wenz, Lena-Sophie; Ellenrieder, Lars; Qiu, Jian; Bohnert, Maria; Zufall, Nicole; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

2015-09-28

Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM-SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane. © 2015 Wenz et al.

Tzfp represses the androgen receptor in mouse testis.

PubMed

Furu, Kari; Klungland, Arne

2013-01-01

The testis zinc finger protein (Tzfp), also known as Repressor of GATA, belongs to the BTB/POZ zinc finger family of transcription factors and is thought to play a role in spermatogenesis due to its remarkably high expression in testis. Despite many attempts to find the in vivo role of the protein, the molecular function is still largely unknown. Here, we address this issue using a novel mouse model with a disrupted Tzfp gene. Homozygous Tzfp null mice are born at reduced frequency but appear viable and fertile. Sertoli cells in testes lacking Tzfp display an increase in Androgen Receptor (AR) signaling, and several genes in the testis, including Gata1, Aie1 and Fanc, show increased expression. Our results indicate that Tzfp function as a transcriptional regulator and that loss of the protein leads to alterations in AR signaling and reduced number of apoptotic cells in the testicular tubules.

Biogenesis of cytosolic ribosomes requires the essential iron–sulphur protein Rli1p and mitochondria

PubMed Central

Kispal, Gyula; Sipos, Katalin; Lange, Heike; Fekete, Zsuzsanna; Bedekovics, Tibor; Janáky, Tamás; Bassler, Jochen; Aguilar Netz, Daili J; Balk, Janneke; Rotte, Carmen; Lill, Roland

2005-01-01

Mitochondria perform a central function in the biogenesis of cellular iron–sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p. PMID:15660134

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: close structural resemblance to E. coli pantothenate kinase.

PubMed

Li de La Sierra-Gallay, Ines; Collinet, Bruno; Graille, Marc; Quevillon-Cheruel, Sophie; Liger, Dominique; Minard, Philippe; Blondeau, Karine; Henckes, Gilles; Aufrère, Robert; Leulliot, Nicolas; Zhou, Cong-Zhao; Sorel, Isabelle; Ferrer, Jean-Luc; Poupon, Anne; Janin, Joël; van Tilbeurgh, Herman

2004-03-01

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of beta-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Copyright 2004 Wiley-Liss, Inc.

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

PubMed

Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

2016-04-05

G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

An archaeal genomic signature

NASA Technical Reports Server (NTRS)

Graham, D. E.; Overbeek, R.; Olsen, G. J.; Woese, C. R.

2000-01-01

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

Genetic Dissection of DivIVA Functions in Listeria monocytogenes.

PubMed

Kaval, Karan Gautam; Hauf, Samuel; Rismondo, Jeanine; Hahn, Birgitt; Halbedel, Sven

2017-12-15

DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future. IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins. Copyright © 2017 American Society for Microbiology.

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

PubMed Central

Grainger, Richard J.; Barrass, J. David; Jacquier, Alain; Rain, Jean-Christophe; Beggs, Jean D.

2009-01-01

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors—namely, Prp8p and Snu114p—and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans. PMID:19854871

Blood coagulation reactions on nanoscale membrane surfaces

NASA Astrophysics Data System (ADS)

Pureza, Vincent S.

Blood coagulation requires the assembly of several membrane-bound protein complexes composed of regulatory and catalytic subunits. The biomembranes involved in these reactions not only provide a platform for these procoagulant proteins, but can also affect their function. Increased exposure of acidic phospholipids on the outer leaflet of the plasma membrane can dramatically modulate the catalytic efficiencies of such membrane-bound enzymes. Under physiologic conditions, however, these phospholipids spontaneously cluster into a patchwork of membrane microdomains upon which membrane binding proteins may preferentially assemble. As a result, the membrane composition surrounding these proteins is largely unknown. Through the development and use of a nanometer-scale bilayer system that provides rigorous control of the phospholipid membrane environment, I investigated the role of phosphatidylserine, an acidic phospholipid, in the direct vicinity (within nanometers) of two critical membrane-bound procoagulant protein complexes and their respective natural substrates. Here, I present how the assembly and function of the tissue factor˙factor VIIa and factor Va˙factor Xa complexes, the first and final cofactor˙enzyme complexes of the blood clotting cascade, respectively, are mediated by changes in their immediate phospholipid environments.

Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein.

PubMed

Chen, Huiqing; Li, Mei; Huang, Guoping; Mai, Weijun; Chen, Keping; Zhou, Yajing

2014-08-01

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

Mutations in the Treacher Collins syndrome gene lead to mislocalization of the nucleolar protein treacle.

PubMed

Marsh, K L; Dixon, J; Dixon, M J

1998-10-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS gene ( TCOF1 ), which is localized to chromosome 5q32-q33.1, recently has been identified by positional cloning. Analysis of TCOF1 revealed that the majority of TCS mutations result in the creation of a premature termination codon. The function of the predicted protein, treacle, is unknown, although indirect evidence from database analyses suggests that it may function as a shuttling nucleolar phosphoprotein. In the current study, we provide the first direct evidence that treacle is a nucleolar protein. An antibody generated against treacle shows that it localizes to the nucleolus. Fusion proteins tagged to a green fluorescent protein reporter were shown to localize to different compartments of the cell when putative nuclear localization signals were deleted. Parallel experiments using conserved regions of the murine homologue of TCOF1 confirmed these results. Site-directed mutagenesis has been used to recreate mutations observed in individuals with TCS. The resulting truncated proteins are mislocalized within the cell, which further supports the hypothesis that an integral part of treacle's function involves shuttling between the nucleolus and the cytoplasm. TCS is, therefore, the first Mendelian disorder resulting from mutations which lead to aberrant expression of a nucleolar protein.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

PubMed

Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

2011-03-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase

PubMed Central

VENKITACHALAM, SRIVIDYA; CHUEH, FU-YU; LEONG, KING-FU; PABICH, SAMANTHA; YU, CHAO-LAN

2011-01-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here we report that, among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine–inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identify the positive regulatory phospho-tyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases. PMID:21234523

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the humanmore » protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.« less

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

PubMed

Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

2004-07-01

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Nonparametric density estimation and optimal bandwidth selection for protein unfolding and unbinding data

NASA Astrophysics Data System (ADS)

Bura, E.; Zhmurov, A.; Barsegov, V.

2009-01-01

Dynamic force spectroscopy and steered molecular simulations have become powerful tools for analyzing the mechanical properties of proteins, and the strength of protein-protein complexes and aggregates. Probability density functions of the unfolding forces and unfolding times for proteins, and rupture forces and bond lifetimes for protein-protein complexes allow quantification of the forced unfolding and unbinding transitions, and mapping the biomolecular free energy landscape. The inference of the unknown probability distribution functions from the experimental and simulated forced unfolding and unbinding data, as well as the assessment of analytically tractable models of the protein unfolding and unbinding requires the use of a bandwidth. The choice of this quantity is typically subjective as it draws heavily on the investigator's intuition and past experience. We describe several approaches for selecting the "optimal bandwidth" for nonparametric density estimators, such as the traditionally used histogram and the more advanced kernel density estimators. The performance of these methods is tested on unimodal and multimodal skewed, long-tailed distributed data, as typically observed in force spectroscopy experiments and in molecular pulling simulations. The results of these studies can serve as a guideline for selecting the optimal bandwidth to resolve the underlying distributions from the forced unfolding and unbinding data for proteins.

Differential proteomic analysis of Aspergillus fumigatus morphotypes reveals putative drug targets.

PubMed

Kubitschek-Barreira, Paula H; Curty, Nathalia; Neves, Gabriela W P; Gil, Concha; Lopes-Bezerra, Leila M

2013-01-14

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, an important opportunistic infection for neutropenic patients. The main risk groups are patients with acute leukemia and bone marrow transplantation recipients. The lack of an early diagnostic test together with the limited spectrum of antifungal drugs remains a setback to the successful treatment of this disease. During invasive infection the inhaled fungal conidia enter the morphogenic cycle leading to angioinvasive hyphae. This work aimed to study differentially expressed proteins of A. fumigatus during morphogenesis. To achieve this goal, a 2D-DIGE approach was applied to study surface proteins extractable by reducing agents of two A. fumigatus morphotypes: germlings and hyphae. Sixty-three differentially expressed proteins were identified by MALDI-ToF/MS. We observed that proteins associated with biosynthetic pathways and proteins with multiple functions (miscellaneous) were over-expressed in the early stages of germination, while in hyphae, the most abundant proteins detected were related to metabolic processes or have unknown functions. Among the most interesting proteins regulated during morphogenesis, two putative drug targets were identified, the translational factor, eEF3 and the CipC-like protein. Neither of these proteins are present in mammalian cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Intracellular Electric Field and pH Optimize Protein Localization and Movement

PubMed Central

Cunningham, Jessica; Estrella, Veronica; Lloyd, Mark; Gillies, Robert; Frieden, B. Roy; Gatenby, Robert

2012-01-01

Mammalian cell function requires timely and accurate transmission of information from the cell membrane (CM) to the nucleus (N). These pathways have been intensively investigated and many critical components and interactions have been identified. However, the physical forces that control movement of these proteins have received scant attention. Thus, transduction pathways are typically presented schematically with little regard to spatial constraints that might affect the underlying dynamics necessary for protein-protein interactions and molecular movement from the CM to the N. We propose messenger protein localization and movements are highly regulated and governed by Coulomb interactions between: 1. A recently discovered, radially directed E-field from the NM into the CM and 2. Net protein charge determined by its isoelectric point, phosphorylation state, and the cytosolic pH. These interactions, which are widely applied in elecrophoresis, provide a previously unknown mechanism for localization of messenger proteins within the cytoplasm as well as rapid shuttling between the CM and N. Here we show these dynamics optimize the speed, accuracy and efficiency of transduction pathways even allowing measurement of the location and timing of ligand binding at the CM –previously unknown components of intracellular information flow that are, nevertheless, likely necessary for detecting spatial gradients and temporal fluctuations in ligand concentrations within the environment. The model has been applied to the RAF-MEK-ERK pathway and scaffolding protein KSR1 using computer simulations and in-vitro experiments. The computer simulations predicted distinct distributions of phosphorylated and unphosphorylated components of this transduction pathway which were experimentally confirmed in normal breast epithelial cells (HMEC). PMID:22623963

Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness.

PubMed

Feng, Zhi-Juan; Xu, Sheng-Chun; Liu, Na; Zhang, Gu-Wen; Hu, Qi-Zan; Gong, Ya-Ming

2018-06-01

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, a family of plant-specific proteins, play crucial roles in plant growth and development and stress response. However, systematical information is unknown regarding the TCP gene family in soybean. In the present study, a total of 54 GmTCPs were identified in soybean, which were grouped into 11 groups with the typical TCP conserved domains. Phylogenetic relationship, protein motif and gene structure analyses distinguished the GmTCPs into two homology classes: Class I and Class II. Class II was then differentiated into two subclasses: CIN and CYC/TB1. Unique cis-element number and composition existed in the promoter regions which might be involved in the gene transcriptional regulation of different GmTCPs. Tissue expression analysis demonstrated the diverse spatiotemporal expression profiles of GmTCPs. Furthermore, the interaction protein of one previously functionally unknown TCP protein-GmTCP8 was investigated. Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10). QRT-PCR assays indicated the distinct expression profiles of GmTCPs in response to abiotic stresses (heat, drought and salt) and stress-related signals (abscisic acid, brassinolide, salicylicacid and methyl jasmonate). These results will facilitate to uncover the possible roles of GmTCPs under abiotic stress and hormone signal responses in soybean. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corbin, Cyrielle; Drouet, Samantha; Markulin, Lucija

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8' lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (-)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset ofmore » genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8' linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (-)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.« less

A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.

PubMed

Corbin, Cyrielle; Drouet, Samantha; Markulin, Lucija; Auguin, Daniel; Lainé, Éric; Davin, Laurence B; Cort, John R; Lewis, Norman G; Hano, Christophe

2018-05-01

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8' lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (-)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8' linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (-)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

PubMed

Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

2017-07-25

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

PubMed Central

Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.

2004-01-01

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290

The Myxococcus xanthus Spore Cuticula Protein C Is a Fragment of FibA, an Extracellular Metalloprotease Produced Exclusively in Aggregated Cells

PubMed Central

Lee, Bongsoo; Mann, Petra; Grover, Vidhi; Treuner-Lange, Anke; Kahnt, Jörg; Higgs, Penelope I.

2011-01-01

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns. PMID:22174937

Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway.

PubMed

Park, Arnold; Yun, Tatyana; Vigant, Frederic; Pernet, Olivier; Won, Sohui T; Dawes, Brian E; Bartkowski, Wojciech; Freiberg, Alexander N; Lee, Benhur

2016-05-01

The budding of Nipah virus, a deadly member of the Henipavirus genus within the Paramyxoviridae, has been thought to be independent of the host ESCRT pathway, which is critical for the budding of many enveloped viruses. This conclusion was based on the budding properties of the virus matrix protein in the absence of other virus components. Here, we find that the virus C protein, which was previously investigated for its role in antagonism of innate immunity, recruits the ESCRT pathway to promote efficient virus release. Inhibition of ESCRT or depletion of the ESCRT factor Tsg101 abrogates the C enhancement of matrix budding and impairs live Nipah virus release. Further, despite the low sequence homology of the C proteins of known henipaviruses, they all enhance the budding of their cognate matrix proteins, suggesting a conserved and previously unknown function for the henipavirus C proteins.

Surface Proteins of Gram-Positive Pathogens: Using Crystallography to Uncover Novel Features in Drug and Vaccine Candidates

NASA Astrophysics Data System (ADS)

Baker, Edward N.; Proft, Thomas; Kang, Haejoo

Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

PubMed

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

2016-06-09

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates.

PubMed

Inupakutika, Madhuri A; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A; Onuchic, Jose' N; Azad, Rajeev K; Padilla, Pamela; Mittler, Ron

2017-02-16

NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the "NEET" motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum's CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins.

The Aged Microenvironment Influences Prostate Carcinogenesis

DTIC Science & Technology

2008-12-01

binding protein-like +36 nucleic acid binding Serpinb5 serine (or cysteine) peptidase inhibitor, clade +9 serine-type endopeptidase inhibitor activity...synthase ( phosphatidate +1.9 phosphatidate cytidylyltransferase activity Car1 carbonic anhydrase 1 +1.9 carbonate dehydratase activity;zinc ion...activity Wdr45l Wdr45 like +1.7 acid phosphatase activity;molecular_function unknown Perp PERP, TP53 apoptosis effector +1.7 structural constituent of

Sequence co-evolution gives 3D contacts and structures of protein complexes

PubMed Central

Hopf, Thomas A; Schärfe, Charlotta P I; Rodrigues, João P G L M; Green, Anna G; Kohlbacher, Oliver; Sander, Chris; Bonvin, Alexandre M J J; Marks, Debora S

2014-01-01

Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution. DOI: http://dx.doi.org/10.7554/eLife.03430.001 PMID:25255213

Architecture of the human interactome defines protein communities and disease networks

PubMed Central

Huttlin, Edward L.; Bruckner, Raphael J.; Paulo, Joao A.; Cannon, Joe R.; Ting, Lily; Baltier, Kurt; Colby, Greg; Gebreab, Fana; Gygi, Melanie P.; Parzen, Hannah; Szpyt, John; Tam, Stanley; Zarraga, Gabriela; Pontano-Vaites, Laura; Swarup, Sharan; White, Anne E.; Schweppe, Devin K.; Rad, Ramin; Erickson, Brian K.; Obar, Robert A.; Guruharsha, K.G.; Li, Kejie; Artavanis-Tsakonas, Spyros; Gygi, Steven P.; Harper, J. Wade

2017-01-01

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidation of how genome variation contributes to disease1–3. Here, we present BioPlex 2.0 (Biophysical Interactions of ORFEOME-derived complexes), which employs robust affinity purification-mass spectrometry (AP-MS) methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein coding genes from the human genome, and constitutes the largest such network to date. With >56,000 candidate interactions, BioPlex 2.0 contains >29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering (MCL)5 of interacting proteins identified more than 1300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization. PMID:28514442

Putative function of hypothetical proteins expressed by Clostridium perfringens type A strains and their protective efficacy in mouse model.

PubMed

Alam, Syed Imteyaz; Dwivedi, Pratistha

2016-10-01

The whole genome sequencing and annotation of Clostridium perfringens strains revealed several genes coding for proteins of unknown function with no significant similarities to genes in other organisms. Our previous studies clearly demonstrated that hypothetical proteins CPF_2500, CPF_1441, CPF_0876, CPF_0093, CPF_2002, CPF_2314, CPF_1179, CPF_1132, CPF_2853, CPF_0552, CPF_2032, CPF_0438, CPF_1440, CPF_2918, CPF_0656, and CPF_2364 are genuine proteins of C. perfringens expressed in high abundance. This study explored the putative role of these hypothetical proteins using bioinformatic tools and evaluated their potential as putative candidates for prophylaxis. Apart from a group of eight hypothetical proteins (HPs), a putative function was predicted for the rest of the hypothetical proteins using one or more of the algorithms used. The phylogenetic analysis did not suggest an evidence of a horizontal gene transfer event except for HP CPF_0876. HP CPF_2918 is an abundant extracellular protein, unique to C. perfringens species with maximum strain coverage and did not show any significant match in the database. CPF_2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF_2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF_2918 protein induced antibody production, a mixed Th1 and Th2 kind of response, and provided partial protection to immunized mice in direct C. perfringens challenge. Copyright © 2016 Elsevier B.V. All rights reserved.

Predictive and comparative analysis of Ebolavirus proteins

PubMed Central

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus. PMID:26158395

Periplasmic Proteins of the Extremophile Acidithiobacillus ferrooxidans

PubMed Central

Chi, An; Valenzuela, Lissette; Beard, Simon; Mackey, Aaron J.; Shabanowitz, Jeffrey; Hunt, Donald F.; Jerez, Carlos A.

2015-01-01

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of obtaining energy by oxidizing ferrous iron or sulfur compounds such as metal sulfides. Some of the proteins involved in these oxidations have been described as forming part of the periplasm of this extremophile. The detailed study of the periplasmic components constitutes an important area to understand the physiology and environmental interactions of microorganisms. Proteomics analysis of the periplasmic fraction of A. ferrooxidans ATCC 23270 was performed by using high resolution linear ion trap-FT MS. We identified a total of 131 proteins in the periplasm of the microorganism grown in thiosulfate. When possible, functional categories were assigned to the proteins: 13.8% were transport and binding proteins, 14.6% were several kinds of cell envelope proteins, 10.8% were involved in energy metabolism, 10% were related to protein fate and folding, 10% were proteins with unknown functions, and 26.1% were proteins without homologues in databases. These last proteins are most likely characteristic of A. ferrooxidans and may have important roles yet to be assigned. The majority of the periplasmic proteins from A. ferrooxidans were very basic compared with those of neutrophilic microorganisms such as Escherichia coli, suggesting a special adaptation of the chemolithoautotrophic bacterium to its very acidic environment. The high throughput proteomics approach used here not only helps to understand the physiology of this extreme acidophile but also offers an important contribution to the functional annotation for the available genomes of biomining microorganisms such as A. ferrooxidans for which no efficient genetic systems are available to disrupt genes by procedures such as homologous recombination. PMID:17911085

Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian

2011-01-01

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80%more » of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.« less

Predictive and comparative analysis of Ebolavirus proteins.

PubMed

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

SMALL ACIDIC PROTEIN1 acts with RUB modification components, the COP9 signalosome, and AXR1 to regulate growth and development of Arabidopsis.

PubMed

Nakasone, Akari; Fujiwara, Masayuki; Fukao, Yoichiro; Biswas, Kamal Kanti; Rahman, Abidur; Kawai-Yamada, Maki; Narumi, Issay; Uchimiya, Hirofumi; Oono, Yutaka

2012-09-01

Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.

Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *

PubMed Central

McDermott, Suzanne M.; Guo, Xuemin; Carnes, Jason; Stuart, Kenneth

2015-01-01

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3′-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages. PMID:26304125

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

PubMed Central

Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

2015-01-01

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

Succinyl-proteome profiling of Dendrobium officinale, an important traditional Chinese orchid herb, revealed involvement of succinylation in the glycolysis pathway.

PubMed

Feng, Shangguo; Jiao, Kaili; Guo, Hong; Jiang, Mengyi; Hao, Juan; Wang, Huizhong; Shen, Chenjia

2017-08-10

Lysine succinylation is a ubiquitous and important protein post-translational modification in various eukaryotic and prokaryotic cells. However, its functions in Dendrobium officinale, an important traditional Chinese orchid herb with high polysaccharide contents, are largely unknown. In our study, LC-MS/MS was used to identify the peptides that were enriched by immune-purification with a high-efficiency succinyl-lysine antibody. In total, 314 lysine succinylation sites in 207 proteins were identified. A gene ontology analysis showed that these proteins are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Moreover, two types of conserved succinylation motifs, '***K suc ******K**' and '****EK suc ***', were identified. Our data showed that lysine succinylation occurred on five key enzymes in the glycolysis pathway. The numbers of average succinylation sites on these five enzymes in plants were lower than those in bacteria and mammals. Interestingly, two active site amino acids residues, K103 and K225, could be succinylated in fructose-bisphosphate aldolase, indicating a potential function of lysine succinylation in the regulation of glycolytic enzyme activities. Furthermore, the protein-protein interaction network for the succinylated proteins showed that several functional terms, such as glycolysis, TCA cycle, oxidative phosphorylation and ribosome, are consisted. Our results provide the first comprehensive view of the succinylome of D. officinale and may accelerate future biological investigations of succinylation in the synthesis of polysaccharides, which are major active ingredients.

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation*

PubMed Central

Netz, Daili J. A.; Pierik, Antonio J.; Stümpfig, Martin; Bill, Eckhard; Sharma, Anil K.; Pallesen, Leif J.; Walden, William E.; Lill, Roland

2012-01-01

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo. PMID:22362766

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Functional and Structural Characterization of Zebrafish ASC.

PubMed

Li, Yajuan; Huang, Yi; Cao, Xiaocong; Yin, Xueying; Jin, Xiangyu; Liu, Sheng; Jiang, Jiansheng; Jiang, Wei; Xiao, Tsan Sam; Zhou, Rongbin; Cai, Gang; Hu, Bing; Jin, Tengchuan

2018-05-23

The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)) is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein (MBP). Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Phyllosphere Metaproteomes of Trees from the Brazilian Atlantic Forest Show High Levels of Functional Redundancy.

PubMed

Lambais, M R; Barrera, S E; Santos, E C; Crowley, D E; Jumpponen, A

2017-01-01

The phyllosphere of the Brazilian Atlantic Forest has been estimated to contain several million bacterial species that are associated with approximately 20000 plant species. Despite the high bacterial diversity in the phyllosphere, the function of these microorganisms and the mechanisms driving their community assembly are largely unknown. In this study, we characterized the bacterial communities in the phyllospheres of four tree species of the Atlantic Forest (Mollinedia schottiana, Ocotea dispersa, Ocotea teleiandra, and Tabebuia serratifolia) and their metaproteomes to examine the basic protein functional groups expressed in the phyllosphere. Bacterial community analyses using 16S rRNA gene sequencing confirmed prior observations that plant species harbor distinct bacterial communities and that plants of the same taxon have more similar communities than more distantly related taxa. Using LC-ESI-Q-TOF, we identified 216 nonredundant proteins, based on 3503 peptide mass spectra. Most protein families were shared among the phyllosphere communities, suggesting functional redundancy despite differences in the species compositions of the bacterial communities. Proteins involved in glycolysis and anaerobic carbohydrate metabolism, solute transport, protein metabolism, cell motility, stress and antioxidant responses, nitrogen metabolism, and iron homeostasis were among the most frequently detected. In contrast to prior studies on crop plants and Arabidopsis, a low abundance of OTUs related to Methylobacterium and no proteins associated with the metabolism of one-carbon molecules were detected in the phyllospheres of the tree species studied here. Our data suggest that even though the phyllosphere bacterial communities of different tree species are phylogenetically diverse, their metaproteomes are functionally convergent with respect to traits required for survival on leaf surfaces.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Olfactory behavior and physiology are disrupted in prion protein knockout mice.

PubMed

Le Pichon, Claire E; Valley, Matthew T; Polymenidou, Magdalini; Chesler, Alexander T; Sagdullaev, Botir T; Aguzzi, Adriano; Firestein, Stuart

2009-01-01

The prion protein PrP(C) is infamous for its role in disease, but its normal physiological function remains unknown. Here we found a previously unknown behavioral phenotype of Prnp(-/-) mice in an odor-guided task. This phenotype was manifest in three Prnp knockout lines on different genetic backgrounds, which provides strong evidence that the phenotype is caused by a lack of PrP(C) rather than by other genetic factors. Prnp(-/-) mice also showed altered behavior in a second olfactory task, suggesting that the phenotype is olfactory specific. Furthermore, PrP(C) deficiency affected oscillatory activity in the deep layers of the main olfactory bulb, as well as dendrodendritic synaptic transmission between olfactory bulb granule and mitral cells. Notably, both the behavioral and electrophysiological alterations found in Prnp(-/-) mice were rescued by transgenic neuronal-specific expression of PrP(C). These data suggest that PrP(C) is important in the normal processing of sensory information by the olfactory system.

Amyotrophic lateral sclerosis: A 40-year personal perspective.

PubMed

Eisen, Andrew

2009-04-01

Amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND) shares with other neurodegenetrative disorders of the aging nervous system a polygenic, multifactorial aetiology. Less than 10% are familial and these too probably are associated with several interactive genes. The onset of ALS predates development of clinical symptoms by an unknown interval which may extend several years. The cause of neurodegeneration remains unknown but a common end-point is protein misfolding which in turn causes cell function failure. The complex nature of ALS has hindered therapeutic advances. In recent years longer survival is attributable largely to institution of non-invasive ventilation with BiPAP and timely implementation of percutaneous endoscopic gastrostomy (PEG) feeding. Symptomatic treatment has advanced improving quality of life. Several encouraging avenues of therapy for ALS are beginning to be emerge raising hope for real benefit. They include protective autoimmunity, vaccines against misfolded protein epitopes and other deleterious species, new drug delivery systems employing nanotechnology and the potential of stem cell therapy.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

PubMed

Brodsky, V Y; Malchenko, L A; Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2016-08-01

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

2013-11-01

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

PubMed Central

Kang, Hong-Jun; Vassilopoulos, Athanassios

2016-01-01

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context. PMID:26966987

Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans

PubMed Central

van der Meer-van Kraaij, Cindy; Siezen, Roland; Kramer, Evelien; Reinders, Marjolein; Blokzijl, Hans; van der Meer, Roelof

2007-01-01

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive “top” face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents. PMID:18850182

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

PubMed Central

Spatuzza, Carmen; Schiavone, Marco; Di Salle, Emanuela; Janda, Elzbieta; Sardiello, Marco; Fiume, Giuseppe; Fierro, Olga; Simonetta, Marco; Argiriou, Notis; Faraonio, Raffaella; Capparelli, Rosanna; Quinto, Ileana

2008-01-01

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk. PMID:18596081

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

PubMed

Rahaman, Siti Nurulnabila A; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

2016-03-01

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

PubMed

Reuter, T; Herterich, S; Bernhard, O; Hoehn, H; Gross, H J

2000-01-15

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

Corticosteroid-Induced MKP-1 Represses Pro-Inflammatory Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM Cells.

PubMed

Prabhala, Pavan; Bunge, Kristin; Ge, Qi; Ammit, Alaina J

2016-10-01

Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti-inflammatory pharmacotherapies, including corticosteroids, are front-line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid-inducible gene, mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP-1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein-tristetraprolin (TTP). But whether corticosteroid-induced MKP-1 acts via p38 MAPK-mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK-dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro-asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL-6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP-1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone-induced MKP-1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro-inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153-2158, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample

NASA Astrophysics Data System (ADS)

Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G. Julius

2016-09-01

Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the ‘footprint’ of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

PubMed

Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

2018-03-26

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana*

PubMed Central

Kotzsch, Alexander; Pawolski, Damian; Milentyev, Alexander; Shevchenko, Anna; Scheffel, André; Poulsen, Nicole; Shevchenko, Andrej; Kröger, Nils

2016-01-01

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis. PMID:26710847

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)

PubMed Central

Bayfield, Mark A.; Yang, Ruiqing; Maraia, Richard J.

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3’OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3’OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3’OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNA assembly by hLARP7/PIP7S). Analyses of other LARP family members (i.e., hLARP4, hLARP6) suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs. PMID:20138158

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs).

PubMed

Bayfield, Mark A; Yang, Ruiqing; Maraia, Richard J

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Aldolase sequesters WASP and affects WASP/Arp2/3-stimulated actin dynamics.

PubMed

Ritterson Lew, Carolyn; Tolan, Dean R

2013-08-01

In addition to its roles in sugar metabolism, fructose-1,6-bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed "moonlighting functions." These moonlighting functions likely involve the known aldolase-actin interaction, as many proteins with which aldolase interacts are involved in actin-dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott-Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP-dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3-dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP-dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP-dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin-binding activity. While the actin-binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP-mediated processes. Copyright © 2013 Wiley Periodicals, Inc.

S-Nitrosylation of Cofilin-1 Mediates Estradiol-17β-Stimulated Endothelial Cytoskeleton Remodeling

PubMed Central

Zhang, Hong-hai; Lechuga, Thomas J.; Tith, Tevy; Wang, Wen; Wing, Deborah A.

2015-01-01

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNOCys80 of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling. PMID:25635941

Real-Time Ligand Binding Pocket Database Search Using Local Surface Descriptors

PubMed Central

Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

2010-01-01

Due to the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of a particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two dimensional pseudo-Zernike moments or the 3D Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark study employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed. PMID:20455259

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

PubMed Central

Eshghi, Azad; Pappalardo, Elisa; Hester, Svenja; Thomas, Benjamin; Pretre, Gabriela

2015-01-01

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins. PMID:25987703

Real-time ligand binding pocket database search using local surface descriptors.

PubMed

Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

2010-07-01

Because of the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two-dimensional pseudo-Zernike moments or the three-dimensional Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark studies employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed.

Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins

PubMed Central

Vanhoutteghem, Amandine; Maciejewski-Duval, Anna; Bouche, Cyril; Delhomme, Brigitte; Hervé, Françoise; Daubigney, Fabrice; Soubigou, Guillaume; Araki, Masatake; Araki, Kimi; Yamamura, Ken-ichi; Djian, Philippe

2009-01-01

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2. PMID:19706529

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

PubMed Central

Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-01-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

PubMed

Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-03-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.

PubMed

Bekliz, Meriem; Azza, Said; Seligmann, Hervé; Decloquement, Philippe; Raoult, Didier; La Scola, Bernard

2018-05-15

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes. Copyright © 2018 American Society for Microbiology.

Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

PubMed

Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

2016-11-01

The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1

PubMed Central

Selb, Regina; Derntl, Christian; Klein, Reinhard; Alte, Beatrix; Hofbauer, Christoph; Kaufmann, Martin; Beraha, Judith; Schöner, Léa

2017-01-01

ABSTRACT In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea. By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadii. IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii. Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression. PMID:28202757

Plasmodesmata: channels for intercellular signaling during plant growth and development.

PubMed

Sevilem, Iris; Yadav, Shri Ram; Helariutta, Ykä

2015-01-01

Plants have evolved strategies for short- and long-distance communication to coordinate plant development and to adapt to changing environmental conditions. Plasmodesmata (PD) are intercellular nanochannels that provide an effective pathway for both selective and nonselective movement of various molecules that function in diverse biological processes. Numerous non-cell-autonomous proteins (NCAP) and small RNAs have been identified that have crucial roles in cell fate determination and organ patterning during development. Both the density and aperture size of PD are developmentally regulated, allowing formation of spatial symplastic domains for establishment of tissue-specific developmental programs. The PD size exclusion limit (SEL) is controlled by reversible deposition of callose, as well as by some PD-associated proteins. Although a large number of PD-associated proteins have been identified, many of their functions remain unknown. Despite the fact that PD are primarily membranous structures, surprisingly very little is known about their lipid composition. Thus, future studies in PD biology will provide deeper insights into the high-resolution structure and tightly regulated functions of PD and the evolution of PD-mediated cell-to-cell communication in plants.

Copper attachment to prion protein at a non-octarepeat site

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Bernholc, Jerry

2011-03-01

Prion protein (PrP) plays a causative role in a group of neurodegenerative diseases, which include ``mad cow disease'' or its human form variant Creutzfeld-Jacob disease. Normal function of PrP remains unknown, but it is now well established that PrP can efficiently bind copper ions and this ability has been linked to its function. The primary binding sites are located in the so-called octarepeat region located between residues 60-91. While these are by now well characterized, the sites located outside these region remain mostly undetermined. In this work, we investigate the properties of Cu binding site located at His 111 using recently developed hybrid Kohn-Sham/orbital-free density functional simulations. Experimental data indicate that copper is coordinated by either four nitrogens or three nitrogens and one oxygen. We investigate both possibilities, comparing their energetics and attachment geometries. Similarities and differences with other binding sites and implications for PrP function will also be discussed.

The multiple functions of plant serine protease inhibitors

PubMed Central

Giri, Ashok P; Kaur, Harleen; Baldwin, Ian T

2011-01-01

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development. PMID:22004998

Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis

PubMed Central

Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K.; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G.

2015-01-01

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes. PMID:25795214

Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus.

PubMed

O'Keeffe, Grainne; Jöchl, Christoph; Kavanagh, Kevin; Doyle, Sean

2013-11-01

The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed. © 2013 The Protein Society.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

PubMed

Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

2016-07-01

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Precursor-product discrimination by La protein during tRNA metabolism.

PubMed

Bayfield, Mark A; Maraia, Richard J

2009-04-01

La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

UV-triggered Affinity Capture Identifies Interactions between the Plasmodium falciparum Multidrug Resistance Protein 1 (PfMDR1) and Antimalarial Agents in Live Parasitized Cells*

PubMed Central

Brunner, Ralf; Ng, Caroline L.; Aissaoui, Hamed; Akabas, Myles H.; Boss, Christoph; Brun, Reto; Callaghan, Paul S.; Corminboeuf, Olivier; Fidock, David A.; Frame, Ithiel J.; Heidmann, Bibia; Le Bihan, Amélie; Jenö, Paul; Mattheis, Corinna; Moes, Suzette; Müller, Ingrid B.; Paguio, Michelle; Roepe, Paul D.; Siegrist, Romain; Voss, Till; Welford, Richard W. D.; Wittlin, Sergio; Binkert, Christoph

2013-01-01

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615. PMID:23754276

EFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijs, Laurence de; Lakaye, Bernard; Coumans, Bernard

2006-09-10

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1more » is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.« less

Rule Mining Techniques to Predict Prokaryotic Metabolic Pathways.

PubMed

Saidi, Rabie; Boudellioua, Imane; Martin, Maria J; Solovyev, Victor

2017-01-01

It is becoming more evident that computational methods are needed for the identification and the mapping of pathways in new genomes. We introduce an automatic annotation system (ARBA4Path Association Rule-Based Annotator for Pathways) that utilizes rule mining techniques to predict metabolic pathways across wide range of prokaryotes. It was demonstrated that specific combinations of protein domains (recorded in our rules) strongly determine pathways in which proteins are involved and thus provide information that let us very accurately assign pathway membership (with precision of 0.999 and recall of 0.966) to proteins of a given prokaryotic taxon. Our system can be used to enhance the quality of automatically generated annotations as well as annotating proteins with unknown function. The prediction models are represented in the form of human-readable rules, and they can be used effectively to add absent pathway information to many proteins in UniProtKB/TrEMBL database.

A molecular imaging analysis of C×43 association with Cdo during skeletal myoblast differentiation

NASA Astrophysics Data System (ADS)

Nosi, Daniele; Mercatelli, Raffaella; Chellini, Flaminia; Soria, Silvia; Pini, Alessandro; Formigli, Lucia; Quercioli, Franco

2014-02-01

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects are independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development

PubMed Central

Wang, Ying; Ren, Yulong; Zhou, Kunneng; Liu, Linglong; Wang, Jiulin; Xu, Yang; Zhang, Huan; Zhang, Long; Feng, Zhiming; Wang, Liwei; Ma, Weiwei; Wang, Yunlong; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Cheng, Zhijun; Wan, Jianmin

2017-01-01

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice. PMID:28694820

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

PubMed

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

PubMed

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

PubMed Central

Williams, Corey L.; Winkelbauer, Marlene E.; Schafer, Jenny C.; Michaud, Edward J.

2008-01-01

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS. PMID:18337471

A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae.

PubMed

Pineda-Lucena, Antonio; Liao, Jack C C; Cort, John R; Yee, Adelinda; Kennedy, Michael A; Edwards, Aled M; Arrowsmith, Cheryl H

2003-05-01

As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.

Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

PubMed Central

Dailing, Angela; Luchini, Alessandra; Liotta, Lance

2016-01-01

Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting, are compared and contrasted. PMID:26400464

The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

PubMed Central

Herzog, Etienne; Guerra-Peraza, Orlene; Hohn, Thomas

2000-01-01

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly. PMID:10666237

Incorporating molecular and functional context into the analysis and prioritization of human variants associated with cancer

PubMed Central

Peterson, Thomas A; Nehrt, Nathan L; Park, DoHwan

2012-01-01

Background and objective With recent breakthroughs in high-throughput sequencing, identifying deleterious mutations is one of the key challenges for personalized medicine. At the gene and protein level, it has proven difficult to determine the impact of previously unknown variants. A statistical method has been developed to assess the significance of disease mutation clusters on protein domains by incorporating domain functional annotations to assist in the functional characterization of novel variants. Methods Disease mutations aggregated from multiple databases were mapped to domains, and were classified as either cancer- or non-cancer-related. The statistical method for identifying significantly disease-associated domain positions was applied to both sets of mutations and to randomly generated mutation sets for comparison. To leverage the known function of protein domain regions, the method optionally distributes significant scores to associated functional feature positions. Results Most disease mutations are localized within protein domains and display a tendency to cluster at individual domain positions. The method identified significant disease mutation hotspots in both the cancer and non-cancer datasets. The domain significance scores (DS-scores) for cancer form a bimodal distribution with hotspots in oncogenes forming a second peak at higher DS-scores than non-cancer, and hotspots in tumor suppressors have scores more similar to non-cancers. In addition, on an independent mutation benchmarking set, the DS-score method identified mutations known to alter protein function with very high precision. Conclusion By aggregating mutations with known disease association at the domain level, the method was able to discover domain positions enriched with multiple occurrences of deleterious mutations while incorporating relevant functional annotations. The method can be incorporated into translational bioinformatics tools to characterize rare and novel variants within large-scale sequencing studies. PMID:22319177

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

2016-08-01

Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2~Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots.

PubMed

Ward, Carl C; Kleinman, Jordan I; Nomura, Daniel K

2017-06-16

Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.

Structure-Based Annotation of a Novel Sugar Isomerase from the Pathogenic E. coli O157:H7

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Staalduinen, L.; Park, C; Yeom, S

2010-01-01

Prokaryotes can use a variety of sugars as carbon sources in order to provide a selective survival advantage. The gene z5688 found in the pathogenic Escherichia coli O157:H7 encodes a 'hypothetical' protein of unknown function. Sequence analysis identified the gene product as a putative member of the cupin superfamily of proteins, but no other functional information was known. We have determined the crystal structure of the Z5688 protein at 1.6 {angstrom} resolution and identified the protein as a novel E. coli sugar isomerase (EcSI) through overall fold analysis and secondary-structure matching. Extensive substrate screening revealed that EcSI is capable ofmore » acting on D-lyxose and D-mannose. The complex structure of EcSI with fructose allowed the identification of key active-site residues, and mutagenesis confirmed their importance. The structure of EcSI also suggested a novel mechanism for substrate binding and product release in a cupin sugar isomerase. Supplementation of a nonpathogenic E. coli strain with EcSI enabled cell growth on the rare pentose d-lyxose.« less

Etomoxir-induced increase in UCP3 supports a role of uncoupling protein 3 as a mitochondrial fatty acid anion exporter.

PubMed

Schrauwen, Patrick; Hinderling, Vera; Hesselink, Matthijs K C; Schaart, Gert; Kornips, Esther; Saris, Wim H M; Westerterp-Plantenga, Margriet; Langhans, Wolfgang

2002-10-01

The physiological function of human uncoupling protein-3 is still unknown. Uncoupling protein-3 is increased during fasting and high-fat feeding. In these situations the availability of fatty acids to the mitochondria exceeds the capacity to metabolize fatty acids, suggesting a role for uncoupling protein-3 in handling of non-metabolizable fatty acids. To test the hypothesis that uncoupling protein-3 acts as a mitochondrial exporter of non-metabolizable fatty acids from the mitochondrial matrix, we gave human subjects Etomoxir (which blocks mitochondrial entry of fatty acids) or placebo in a cross-over design during a 36-h stay in a respiration chamber. Etomoxir inhibited 24-h fat oxidation and fat oxidation during exercise by approximately 14-19%. Surprisingly, uncoupling protein-3 content in human vastus lateralis muscle was markedly up-regulated within 36 h of Etomoxir administration. Up-regulation of uncoupling protein-3 was accompanied by lowered fasting blood glucose and increased translocation of glucose transporter-4. These data support the hypothesis that the physiological function of uncoupling protein-3 is to facilitate the outward transport of non-metabolizable fatty acids from the mitochondrial matrix and thus prevents mitochondria from the potential deleterious effects of high fatty acid levels. In addition our data show that up-regulation of uncoupling protein-3 can be beneficial in the treatment of type 2 diabetes.

Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

PubMed Central

Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M.; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

2016-01-01

Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

A Common Fold Mediates Vertebrate Defense and Bacterial Attack

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosado, Carlos J.; Buckle, Ashley M.; Law, Ruby H.P.

2008-10-02

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterialmore » and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.« less

In-Depth N-Glycosylation Reveals Species-Specific Modifications and Functions of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana).

PubMed

Feng, Mao; Fang, Yu; Han, Bin; Xu, Xiang; Fan, Pei; Hao, Yue; Qi, Yuping; Hu, Han; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

2015-12-04

Royal jelly (RJ), secreted by honeybee workers, plays diverse roles as nutrients and defense agents for honeybee biology and human health. Despite being reported to be glycoproteins, the glycosylation characterization and functionality of RJ proteins in different honeybee species are largely unknown. An in-depth N-glycoproteome analysis and functional assay of RJ produced by Apis mellifera lingustica (Aml) and Apis cerana cerana (Acc) were conducted. RJ produced by Aml yielded 80 nonredundant N-glycoproteins carrying 190 glycosites, of which 23 novel proteins harboring 35 glycosites were identified. For Acc, all 43 proteins glycosylated at 138 glycosites were reported for the first time. Proteins with distinct N-glycoproteomic characteristics in terms of glycoprotein species, number of N-glycosylated sites, glycosylation motif, abundance level of glycoproteins, and N-glycosites were observed in this two RJ samples. The fact that the low inhibitory efficiency of N-glycosylated major royal jelly protein 2 (MRJP2) against Paenibacillus larvae (P. larvae) and the absence of antibacterial related glycosylated apidaecin, hymenoptaecin, and peritrophic matrix in the Aml RJ compared to Acc reveal the mechanism for why the Aml larvae are susceptible to P. larvae, the causative agent of a fatal brood disease (American foulbrood, AFB). The observed antihypertension activity of N-glycosylated MRJP1 in two RJ samples and a stronger activity found in Acc than in Aml reveal that specific RJ protein and modification are potentially useful for the treatment of hypertensive disease for humans. Our data gain novel understanding that the western and eastern bees have evolved species-specific strategies of glycosylation to fine-tune protein activity for optimizing molecular function as nutrients and immune agents for the good of honeybee and influence on the health promoting activity for human as well. This serves as a valuable resource for the targeted probing of the biological functions of RJ proteins for honeybee and medical communities.

MoCha: Molecular Characterization of Unknown Pathways.

PubMed

Lobo, Daniel; Hammelman, Jennifer; Levin, Michael

2016-04-01

Automated methods for the reverse-engineering of complex regulatory networks are paving the way for the inference of mechanistic comprehensive models directly from experimental data. These novel methods can infer not only the relations and parameters of the known molecules defined in their input datasets, but also unknown components and pathways identified as necessary by the automated algorithms. Identifying the molecular nature of these unknown components is a crucial step for making testable predictions and experimentally validating the models, yet no specific and efficient tools exist to aid in this process. To this end, we present here MoCha (Molecular Characterization), a tool optimized for the search of unknown proteins and their pathways from a given set of known interacting proteins. MoCha uses the comprehensive dataset of protein-protein interactions provided by the STRING database, which currently includes more than a billion interactions from over 2,000 organisms. MoCha is highly optimized, performing typical searches within seconds. We demonstrate the use of MoCha with the characterization of unknown components from reverse-engineered models from the literature. MoCha is useful for working on network models by hand or as a downstream step of a model inference engine workflow and represents a valuable and efficient tool for the characterization of unknown pathways using known data from thousands of organisms. MoCha and its source code are freely available online under the GPLv3 license.

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo¶

PubMed Central

Deterding, Leesa J.; Williams, Jason G.; Humble, Margaret M.; Petrovich, Robert M.; Wei, Sung-Jen; Trempus, Carol S.; Gates, Matthew B.; Zhu, Feng; Smart, Robert C.; Tennant, Raymond W.; Tomer, Kenneth B.

2010-01-01

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34. PMID:21499536

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

PubMed Central

Castellano, Joseph M.; Mosher, Kira I.; Abbey, Rachelle J.; McBride, Alisha A.; James, Michelle L.; Berdnik, Daniela; Shen, Jadon C.; Zou, Bende; Xie, Xinmin S.; Tingle, Martha; Hinkson, Izumi V.; Angst, Martin S.; Wyss-Coray, Tony

2017-01-01

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation1, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation2, 3, 4 and downregulated in the aged brain5, 6, 7, 8. In addition to revitalizing other aged tissues9, 10, 11, 12, 13, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters14, 15, 16, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown17. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction. PMID:28424512

Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates

NASA Astrophysics Data System (ADS)

Inupakutika, Madhuri A.; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A.; Onuchic, Jose' N.; Azad, Rajeev K.; Padilla, Pamela; Mittler, Ron

2017-02-01

NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins.