Carboxyl Terminus Of Protein Research Articles

A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H(2)(18)O or H(2)(16)O, during trypsinization allowed relative quantification of the degree of enrichment of proteins within membranes after phase partitioning with polyethylene glycol/dextran mixtures. The ratios allowed the quantification of 174 membrane-associated proteins with 70 showing plasma membrane enrichment equal to or greater than ATP-dependent proton pumps, canonical plasma membrane proteins. Enriched proteins included several hallmark plasma membrane proteins, such as H(+)-ATPases, aquaporins, receptor-like kinases, and various transporters, as well as a number of proteins with unknown functions. Most importantly, a comparison of the datasets from a sequencing "survey" analysis using the ion trap mass spectrometer with that from the quantitative dual isotope labeling ratio method indicates that as many as one-fourth of the putative survey identifications are biological contaminants rather than bona fide plasma membrane proteins.

A Tryptophan-Rich Motif in the Carboxyl Terminus of the Small Envelope Protein of Hepatitis B Virus Is Central to the Assembly of Hepatitis Delta Virus Particles

The small hepatitis B virus surface antigen (S-HBsAg) is capable of driving the assembly and secretion of hepatitis delta virus (HDV) particles by interacting with the HDV ribonucleoprotein (RNP). Previously, a specific domain of the S-HBsAg protein carboxyl terminus, including a tryptophan residue at position 196 (W196), was proven essential for HDV maturation (S. Jenna and C. Sureau, J. Virol. 73: 3351-3358, 1999). Mutation of W196 to phenylalanine (W196F) was permissive for HBV subviral particle (SVP) secretion but deleterious to HDV virion assembly. Here, the W196F S-HBsAg deficiency was assigned to a loss of its ability for interaction with the large HDV antigen (L-HDAg), a major component of the RNP. Because the overall S-HBsAg carboxyl terminus is particularly rich in tryptophan, an amino acid frequently involved in protein-protein interactions, site-directed mutagenesis was conducted to investigate the function of the S-HBsAg Trp-rich domain in HDV assembly. Single substitutions of tryptophan between positions 163 and 201 with alanine or phenylalanine were tolerated for SVP secretion, but those affecting W196, W199, and W201 were detrimental for HDV assembly. This was proven to result from a reduced capacity of the mutants for interaction with L-HDAg. In addition, a W196S S-HBsAg mutant, which has been described in HBV strains that arose in a few cases of lamivudine-treated HBV-infected patients, was deficient for HDV assembly as a consequence of its impaired capacity for interacting with L-HDAg. Interestingly, the fact that even the most conservative substitution of phenylalanine for tryptophan at positions 196, 199, or 201 was sufficient to ablate interaction of S-HBsAg with L-HDAg suggests that W196, W199, and W201 are located at a binding interface that is central to HDV maturation.

The Carboxyl Terminus of NifK Protein is Involved in Formation of a Stable Nitrogenase Complex under Acidic Growth Conditions in Azotobacter vinelandii

The Carboxyl Terminus of NifK Protein is Involved in Formation of a Stable Nitrogenase Complex under Acidic Growth Conditions in Azotobacter vinelandii

Cysteine String Protein Monitors Late Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.

Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3′ end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.

MITF and cell proliferation: the role of alternative splice forms

Recent studies show that the melanocyte transcription factor MITF not only activates differentiation genes but also genes involved in the regulation of the cell cycle, suggesting that it provides a link between cell proliferation and differentiation. MITF, however, comes in a variety of splice isoforms with potentially distinct biological activities. In particular, there are two isoforms, (-) and (+) MITF, that differ in six residues located upstream of the DNA binding basic domain and show slight differences in the efficiency with which they bind to target DNA. Using in vitro BrdU incorporation assays and FACS analysis in transiently transfected cells, we show that (+) MITF has a strong inhibitory effect on DNA synthesis while (-) MITF has none or only a mild one. The strong inhibitory activity of (+) MITF is not influenced by a number of mutations that modulate MITF's transcriptional activities and is independent of the protein's carboxyl terminus but dependent on its aminoterminus. A further dissection of the molecule points to the importance of an aminoterminal serine, serine-73, which in both isoforms is phosphorylated to comparable degrees. The results suggest that one or several aminoterminal domains cooperate with the alternatively spliced hexapeptide to render MITF anti-proliferative in a way that does not depend on direct E box binding.

A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PP i) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PP i group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (P i), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PP i release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.

Composition and assembly of STAT-targeting ubiquitin ligase complexes: paramyxovirus V protein carboxyl terminus is an oligomerization domain.

Transcription regulators STAT1 and STAT2 are key components of the interferon signaling system leading to innate antiviral immunity. The related STAT3 protein is a regulator of interleukin-6-type cytokine signals and can contribute to both cell growth and death important for cancer gene regulation and tumor survival. These three STAT proteins are targeted for proteasome-mediated degradation by RNA viruses in the Rubulavirus genus of the Paramyxoviridae. A single viral protein, the V protein, assembles STAT-specific ubiquitin ligase complexes from cellular components. Simian virus 5 (SV5) targets STAT1, human parainfluenza virus 2 targets STAT2, and mumps virus targets both STAT1 and STAT3. Analysis of the V-dependent degradation complex (VDC) composition and assembly revealed several features contributing to targeting specificity. SV5 and mumps V proteins require STAT2 to recruit the STAT1 target, yet mumps V protein binds STAT3 independent of STAT1 and STAT2. All Rubulavirus V proteins tested require cellular DDB1 to target STATs for degradation but differ in the use of Roc1, which is essential for mumps V STAT3 targeting. Protein interaction analysis reveals that paramyxovirus V proteins can homo- and heterooligomerize and that the conserved cysteine-rich zinc-binding C-terminal domain is necessary and sufficient for oligomerization. Purified SV5 V protein spontaneously assembles into spherical macromolecular particles, and similar particles constitute SV5 and mumps VDC preparations.

CHIP (Carboxyl Terminus of Hsc70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Estrogen Receptor-α

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.

The Co-chaperone Carboxyl Terminus of Hsp70-interacting Protein (CHIP) Mediates α-Synuclein Degradation Decisions between Proteasomal and Lysosomal Pathways

Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.

Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels

Transient receptor potential channels (TRP) have emerged as cellular sensors of various internal and external cues. Generally, the activation of TRP canonical (TRPC) channels is triggered by the stimulation of phospholipase C; however, multiple factors are involved in the regulation of these channels. Among them, Ca(2+)-mediated feedback channel modulations are often mediated by calmodulin (CaM) and other Ca(2+)-binding proteins. In vitro binding studies have revealed multiple CaM-binding sites on TRPC proteins. Among them, a common CaM/inositol 1,4,5-trisphosphate receptor-binding site is found at the carboxyl terminus of every TRPC isoform. Additional non-conserved CaM-binding sites are present at the amino and carboxyl termini of several TRPC proteins. Likewise, multiple CaM-binding sites were found in other TRP proteins. These, together with the presence in close vicinity of the interaction sites for the related neuronal Ca(2+)-binding proteins, such as CaBP1, suggest a multitude of diverse intracellular Ca(2+)-dependent regulations of TRP channels. Functional studies have begun to reveal the unique roles of CaM and CaBP1 binding to several TRP channels. This review will focus on the CaM- and CaBP1-mediated regulations of TRPC channels. Related studies on TRPM and TRPV channels will also be highlighted.

The carboxyl terminus of the HCV NS5A protein and the 3′-UTR stabilize the expression of NS5B RNA-dependent RNA polymerase in escherichia coli: David N Frick, New York Medical College, Valhala, NY

The carboxyl terminus of the HCV NS5A protein and the 3′-UTR stabilize the expression of NS5B RNA-dependent RNA polymerase in escherichia coli: David N Frick, New York Medical College, Valhala, NY

The Co-chaperone Carboxyl Terminus of Hsp70-interacting Protein (CHIP) Mediates α-Synuclein Degradation Decisions between Proteasomal and Lysosomal Pathways

The Co-chaperone Carboxyl Terminus of Hsp70-interacting Protein (CHIP) Mediates α-Synuclein Degradation Decisions between Proteasomal and Lysosomal Pathways

U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy.

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

The Role of the Carboxyl Terminus in ClC Chloride Channel Function

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.

PDZ domains - common players in the cell signaling.

PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.

The Functions of the N Terminus of the φX174 Internal Scaffolding Protein, a Protein Encoded in an Overlapping Reading Frame in a Two Scaffolding Protein System

φX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5′ deleted genes were constructed and the proteins expressed in vivo. The ΔNH 2 B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.

PDZ Domain Proteins: Plug and Play!

Protein-protein interactions are key elements in building functional protein complexes. Among the plethora of domains identified during the last 10 years, PDZ domains are one of the most commonly found protein-protein interaction domains in organisms from bacteria to humans. Although they may be the sole protein interaction domain within a cytoplasmic protein, they are most often found in combination with other protein interaction domains (for instance, SH3, PTB, WW) participating in complexes that facilitate signaling or determine the localization of receptors. Diversity of PDZ-containing protein function is provided by the large number of PDZ proteins that Mother Nature has distributed in the genome and implicates this protein family in the wiring of a huge number of molecules in molecular networks from the plasma membrane to the nucleus. Although at first sight their binding specificity appeared rather monotonous, involving only binding to the carboxyl-terminus of various proteins, it is now recognized that PDZ domains interact with greater versatility through PDZ-PDZ domain interaction; they bind to internal peptide sequences and even to lipids. Furthermore, PDZ domain-mediated interactions can sometimes be modulated in a dynamic way through target phosphorylation. In this review, we attempt to describe the structural basis of PDZ domain recognition and to give some functional insights into their role in the scaffolding of protein complexes implicated in normal and pathological biological processes.