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Recent studies indicate that the protein affected in spinal muscular atrophy, SMN, plays a role in the assembly of a number of macromolecular complexes that function in the nucleus, interacting with its partner proteins via their arginine- and glycine-rich domains.
Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS). WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN protein regarding its binding partners and the site of protein-protein interaction. The mapping analysis of protein interaction sites in WRN for most of its binding partners have revealed a common site of protein-protein interaction in the RecQ conserved (RQC) region of WRN.
The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.
Sulphate-reducing organisms are widespread in anaerobic enviroments, including the gastrointestinal tract of man and other animals. The study of these bacteria has attracted much attention over the years, due also to the fact that they can have important implications in industry (in biocorrosion and souring of oil and gas deposits), health (in inflamatory bowel diseases) and the environment (bioremediation). The characterization of the various components of the electron transport chain associated with the hydrogen metabolism in Desulfovibrio has generated a large and comprehensive list of studies. This review summarizes the more relevant aspects of the current information available on the structural data of various molecules associated with hydrogen metabolism, namely hydrogenases and cytochromes. The transmembrane redox complexes known to date are also described and discussed. Redox-Bohr and cooperativity effects, observed in a few cytochromes, and believed to be important for their functional role, are discussed. Kinetic studies performed with these redox proteins, showing clues to their functional inter-relationship, are also addressed. These provide the groundwork for the application of a variety of molecular modelling approaches to understanding electron transfer and protein interactions among redox partners, leading to the characterization of several transient periplasmic complexes. In contrast to the detailed understanding of the periplasmic hydrogen oxidation process, very little is known about the cytoplasmic side of the respiratory electron transfer chain, in terms of molecular components (with exception of the terminal reductases), their structure and the protein-protein interactions involved in sulphate reduction. Therefore, a thorough understanding of the sulphate respiratory chain in Desulfovibrio remains a challenging task.
Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.
Entry into mitosis is essentially driven by cyclin B1 and its associated catalytically active partner Cdk1. While cyclin B1 is kept cytoplasmic throughout interphase, nuclear accumulation occurs just prior to mitosis. This restriction is thought to be part of an oscillating mechanism to properly time mitotic entry. A novel nuclear SCF-type mammalian E3 ligase defined by the F-box containing protein NIPA (nuclear interaction partner of ALK), SCFNIPA, targets nuclear cyclin B1 in interphase while it allows for accumulation at G2/M. Thus, oscillating ubiquitination of nuclear cyclin B1 driven by the SCFNIPA complex contributes to the timing of mitotic entry in the mammalian cell cycle.
The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.
Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter the encoded amino acid sequence, and are thus likely to affect the function of the proteins, and represent potential disease-modifiers. There is an enormous number of nsSNPs in the human population, and the major challenge lies in distinguishing the functionally significant and potentially disease-related ones from the rest. In this study, we analyzed the genetic variations that can alter the functions and the interactions of a group of cell cycle proteins (n = 60) and the proteins interacting with them (n = 26) using computational tools. As a result, we extracted 249 nsSNPs from 77 cell cycle proteins and their interaction partners from public SNP databases. Only 31 (12.4%) of the nsSNPs were validated. The majority (64.5%) of the validated SNPs were rare (minor allele frequencies < 5%). Evolutionary conservation analysis using the SIFT tool suggested that 16.1% of the validated nsSNPs may disrupt the protein function. In addition, 58% of the validated nsSNPs were located in functional protein domains/motifs, which together with the evolutionary conservation analysis enabled us to infer possible biological consequences of the nsSNPs in our set. Our study strongly suggests the presence of naturally occurring genetic variations in the cell cycle proteins that may affect their interactions and functions with possible roles in complex human diseases, such as cancer.
We demonstrate that protein-protein interaction networks in several eukaryotic organisms contain significantly more self-interacting proteins than expected if such homodimers randomly appeared in the course of the evolution. We also show that on average homodimers have twice as many interaction partners than non-self-interacting proteins. More specifically, the likelihood of a protein to physically interact with itself was found to be proportional to the total number of its binding partners. These properties of dimers are in agreement with a phenomenological model, in which individual proteins differ from each other by the degree of their 'stickiness' or general propensity toward interaction with other proteins including oneself. A duplication of self-interacting proteins creates a pair of paralogous proteins interacting with each other. We show that such pairs occur more frequently than could be explained by pure chance alone. Similar to homodimers, proteins involved in heterodimers with their paralogs on average have twice as many interacting partners than the rest of the network. The likelihood of a pair of paralogous proteins to interact with each other was also shown to decrease with their sequence similarity. This points to the conclusion that most of interactions between paralogs are inherited from ancestral homodimeric proteins, rather than established de novo after duplication. We finally discuss possible implications of our empirical observations from functional and evolutionary standpoints.
Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.
Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.
Because nitric oxide (NO) may be a ubiquitous regulator of cellular signaling, we have modified the yeast two-hybrid system to explore the possibility of NO-dependent protein-protein interactions. We screened for binding partners of procaspase-3, a protein implicated in apoptotic signaling pathways, and identified multiple NO-dependent interactions.Two such interactions, with acid sphingomyelinase and NO synthase, were shown to occur in mammalian cells dependent on endogenous NO. Nitrosylation may thus provide a broad-based mechanism for regulating interactions between proteins. If so, systematic proteomic analyses in which redox state and NO bioavailability are carefully controlled will reveal a large array of novel interactions.
AtCyp59 and its orthologs from different organisms belong to a family of modular proteins consisting of a peptidyl-prolyl cis-trans isomerase (PPIase) domain, followed by an RNA recognition motif (RRM), and a C-terminal domain enriched in charged amino acids. AtCyp59 was identified in a yeast two-hybrid screen as an interacting partner of the Arabidopsis SR protein SCL33/SR33. The interaction with SCL33/SR33 and with a majority of Arabidopsis SR proteins was confirmed by in vitro pull-down assays. Consistent with these interactions, AtCyp59 localizes to the cell nucleus, but it does not significantly colocalize with SR proteins in nuclear speckles. Rather, it shows a punctuate localization pattern resembling transcription sites. Indeed, by using yeast two-hybrid, in vitro pull-down, and immunoprecipitation assays, we found that AtCyp59 interacts with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Ectopic expression of the tagged protein in Arabidopsis cell suspension resulted in highly reduced growth that is most probably due to reduced phosphorylation of the CTD. Together our data suggest a possible function of AtCyp59 in activities connecting transcription and pre-mRNA processing. We discuss our data in the context of a dynamic interplay between transcription and pre-mRNA processing.
The inhibitor of growth (ING) family of proteins is an evolutionarily conserved family, with members present from yeast to humans. The mammalian ING proteins are candidate tumor suppressor proteins and accordingly can cooperate with p53 to arrest proliferation and induce apoptosis. ING proteins are also reported to function in the promotion of cellular senescence, the regulation of DNA damage responses and the inhibition of angiogenesis. At the molecular level, ING proteins are thought to function as chromatin regulatory molecules, acting as co-factors for distinct histone and factor acetyl-transferase (H/FAT) and deacetylase (HDAC) enzyme complexes. Further, ING proteins interact with a number of additional proteins involved in the regulation of critical nuclear processes, such as gene expression and DNA replication, and also function as nuclear phosphoinositide (PtdInsP) receptors. Despite the increasing number of known molecular interacting partners for ING proteins, the specific biochemical action of mammalian ING proteins and its relationship to tumor suppression remain elusive. In this Prospect, we summarize the present understanding of the binding partners and physiologic roles of ING proteins and propose a general molecular paradigm for how ING proteins might function to prevent cancer.
The recent sequencing of entire eukaryotic genomes has renewed the interest in identifying and characterizing all gene products that are expressed in a given organism. The characterization of unknown gene products is facilitated by the knowledge of its binding partners. Thus, a novel protein may be classified by identifying previously characterized proteins that interact with it. If such an approach is carried out on a large scale, it may allow the rapid characterization of the thousands of predicted open reading frames identified by recent sequencing projects. Currently, the yeast two-hybrid system is the most widely used genetic assay for the detection of protein-protein interactions. The yeast two-hybrid system has become popular because it requires little individual optimization and because, as compared to conventional biochemical methods, the identification and characterization of protein-protein interactions can be completed in a relatively short time span. In this review, we briefly discuss the yeast two-hybrid system and its application to large scale screening studies that aim at deciphering all protein-protein interactions taking place in a given cell type or organism. We then focus on a class of proteins that is unsuitable for conventional yeast two-hybrid systems, namely integral membrane proteins and membrane-associated proteins, and describe several novel genetic systems that combine the advantages of the yeast two-hybrid system with the potential to identify interaction partners of membrane-associated proteins in their natural setting.
The tobacco N gene confers resistance to Tobacco mosaic virus (TMV) and encodes a toll-interleukin-1 receptor/nucleotide binding/Leu-rich repeat class protein. Recent evidence indicates that the Nicotiana benthamiana Rar1 gene (NbRar1), which encodes a protein with a zinc finger motif called CHORD (Cys- and His-rich domain), is required for the function of N. To investigate the role of NbRar1 in plant defense, we identified its interaction partners. We show that the NbRar1 protein interacts with NbSGT1, a highly conserved component of the SCF (Skp1/Cullin/F-box protein)-type E3 ubiquitin ligase complex involved in protein degradation. In addition, we show that NbSGT1 interacts with NbSKP1. Suppression of NbSGT1 and NbSKP1 shows that these genes play an important role in the N-mediated resistance response to TMV. Both NbRar1 and NbSGT1 associate with the COP9 signalosome, another multiprotein complex involved in protein degradation via the ubiquitin-proteasome pathway. Silencing of the NbCOP9 signalosome also compromises N-mediated resistance to TMV. Our results reveal new roles for SCF and the COP9 signalosome in plant defense signaling.
Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2-/- mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.