Identification of a Novel Zinc Metalloprotease through a Global Analysis of Clostridium difficile Extracellular Proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.     

胰岛再生源蛋白(Reg)在组织修复中的功能和调控机制

胰岛再生源蛋白(regenerating islet-derived protein,Reg)是一个多功能分子,在多种生理、病理活动中发挥重要作用。该文主要综述Reg蛋白在组织损伤后促进细胞增殖、抑制炎症因子过表达、调控细胞凋亡和抑制病原微生物生长和扩散的功能及调控机制,为治疗组织损伤提供新思路和新途径。     

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred.     

Olfactomedin Domain-Containing Proteins: Possible Mechanisms of Action and Functions in Normal Development and Pathology

A family of olfactomedin domain-containing proteins consists of at least 13 members in mammals. Although the first protein belonging to this family, olfactomedin, was isolated and partially characterized from frog olfactory neuroepithelim almost 20 years ago, the functions of many family members remain elusive. Most of the olfactomedin domain-containing proteins, similar to frog olfactomedin, are secreted glycoproteins that demonstrate specific expression patterns. Other family members are membrane-bound proteins that may serve as receptors. More than half of the olfactomedin domain-containing genes are expressed in neural tissues. Data obtained over the last several years demonstrate that olfactomedin domain-containing proteins play important roles in neurogenesis, neural crest formation, dorsal ventral patterning, cell–cell adhesion, cell cycle regulation, and tumorigenesis and may serve as modulators of critical signaling pathways (Wnt, bone morphogenic protein). Mutations in two genes encoding myocilin and olfactomedin 2 were implicated in glaucoma, and a growing number of evidence indicate that other genes belonging to the family of olfactomedin domain-containing proteins may contribute to different human disorders including psychiatric disorders. In this review, we summarize recent advances in understanding the possible roles of these proteins with special emphasis on the proteins that are preferentially expressed and function in neural tissues.     

A Disintegrin-like and Metalloprotease (Reprolysin-type) with Thrombospondin Type 1 Motif (ADAMTS) Superfamily: Functions and Mechanisms

Together with seven ADAMTS-like proteins, the 19 mammalian ADAMTS proteases constitute a superfamily. ADAMTS proteases are secreted zinc metalloproteases whose hallmark is an ancillary domain containing one or more thrombospondin type 1 repeats. ADAMTS-like proteins resemble ADAMTS ancillary domains and lack proteolytic activity. Vertebrate expansion of the superfamily reflects emergence of new substrates, duplication of proteolytic activities in new contexts, and cooperative functions of the duplicated genes. ADAMTS proteases are involved in maturation of procollagen and von Willebrand factor, as well as in extracellular matrix proteolysis relating to morphogenesis, angiogenesis, ovulation, cancer, and arthritis. New insights into ADAMTS mechanisms indicate significant regulatory roles for ADAMTS ancillary domains, propeptide processing, and glycosylation. ADAMTS-like proteins appear to have regulatory roles in the extracellular matrix.     

Target Regulation of Axotomy-Sensitive Proteins

Large changes in the production of certain proteins often follow axotomy. How the cell body is signaled to make these changes, or terminate them after regeneration is finished, is unclear. This issue was addressed by studying an axotomized giant identified neuron, the giant cerebral neuron of the sea slug Aplysia, both in vivo and in culture. One week after axon crush in vivo, there were increases of 1.5-18-fold in the 5-h incorporation of [35S]methionine into seven proteins identified by two-dimensional gel electrophoresis. There were decreases of five- to 28-fold in the labeling of four other proteins. An axotomized giant cerebral neuron grows vigorously when placed in culture and forms chemical synapses with appropriate target cells while continuing unabated growth. The labeling of two of the proteins that up-regulate after axotomy in vivo was suppressed by the presence of target cells in culture. For one of the proteins, this effect was also produced by membranes of target cells, but not by medium conditioned by exposure to target cells. These results are consistent with the idea that loss of membrane-membrane contact with target cells (or its restoration) is involved in the initiation (or termination) of the up-regulation of certain proteins after axotomy.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.     

Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

Background

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited.

Methodology/Principal Findings

We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced.

Conclusion/Significance

Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.     

Proteome-Level Analysis Indicates Global Mechanisms for Post-Translational Regulation of RRM Domains

RRM, or RNA-recognition motif, domains are the largest class of single-stranded RNA binding domains in the human proteome and play important roles in RNA processing, splicing, export, stability, packaging, and degradation. Using a current database of post-translational modifications (PTMs), ProteomeScout, we found that RRM domains are also one of the most heavily modified domains in the human proteome. Here, we present two interesting findings about RRM domain modifications, found by mapping known PTMs onto RRM domain alignments and structures. First, we find significant overlap of ubiquitination and acetylation within RRM domains, suggesting the possibility for ubiquitination-acetylation crosstalk. Additionally, an analysis of quantitative study of ubiquitination changes in response to proteasome inhibition highlights the uniqueness of RRM domain ubiquitination – RRM domain ubiquitination decreases in response to proteasome inhibition, whereas the majority of sites increase. Second, we found conservation of tyrosine phosphorylation within the RNP1 and RNP2 consensus sequences, which coordinate RNA binding – suggesting a possible role for regulation of RNA binding by tyrosine kinase signaling. These observations suggest there are unique regulatory mechanisms of RRM function that have yet to be uncovered and that the RRM domain represents a model system for further studies on understanding PTM crosstalk.     

MicroRNAs: Mechanisms, Functions and Progress

In 1993 when Ambros and co-workers [1] discovered that a mysterious Caenorhabditis elegans gene, lin-4, does not encode a protein, but acts in the form of a small RNA and represses the expression of its target gene, lin-14, through base-pairing with its 3 0 untranslated region (3 0 UTR), nobody would imagine that 20 years later,     

Identification of a Proteolytic Bacterium,HW08, and Characterization of Its Extracellular Cold-Active Alkaline Metalloprotease Ps5

A psychrophilic protease-producing bacterium, HW08, was isolated from sediment of the Yellow Sea in eastern China. On the basis of 16S rDNA sequence analysis and physiological properties, the isolate was identified as Pseudomonas lundensis. The secreted protease, named Ps5, was purified from the culture supernatant as a monomer with an apparent molecular mass of 46 kDa on SDS–PAGE. As a metalloprotease (inhibited by EDTA), the enzyme showed maximum activity at 30 °C at pH 10.4. It had no activity loss exposed at 4 °C for 60 d or under repeated freezing and thawing. Broad temperature (25–40 °C) and pH (7.0–11.0) stability was observed in the presence of 5 mm Ca²⁺. Furthermore, the enzyme was resistant to detergent additives such as non-ionic surfactants and bleaches. It showed considerable potential for industry that requires alkaline-protease.     

Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.     

Rab11的结构特征、效应因子与功能

Rab11是Rab小分子GTP酶家族的成员.在细胞内膜泡再循环途径中,Rab11作为重要调节因子,介导膜泡从内体向质膜的运输.近年来随着对Rab11研究的深入,人们发现该蛋白质在多种细胞生命活动中发挥着关键作用.现对Rab11的结构、效应蛋白及功能等方面进行了综述.     

DCAF家族蛋白研究进展

DCAF (DDB1-and CUL4-associated factor)是一类新发现的含"WDXR"结构域的蛋白质家族,其可与CRL4-DDB1构成E3泛素连接酶,通过对靶蛋白进行泛素化修饰,实现对细胞生长、分化、凋亡等一系列生命活动的调控。DCAF蛋白的异常表达通常与肿瘤、发育障碍等疾病密切相关。本文主要根据目前的研究成果对DCAF家族蛋白的功能及机制展开论述。     

Functions and Roles of Proteins: Diabetes as a Paradigm

Molecular and cellular biology has moved towards complete and accurate knowledge of how molecules behave in space and time. Protein is considered as the primary group of molecules responsible for mediating most physiological processes. Changes in the levels of proteins may lead to the altered function and are responsible for many diseases. This review provides a partial molecular explanation of biological force-ratio generation that may act to split protein into branches, and shows molecular functional divergence. Developing a non-reductionist theory of the cellular function in medicine is clearly not sufficient. Finding effective parameters of the models by characterizing molecular interactions becomes necessary. Protein interactivity and stability provides a basis for an integrated understanding of pathologies such diabetes. One example of how a mechanistic analysis of such physiological processes can be of value is the time-delay between mRNA and translation that can act as a fork allowing a slowdown in gene expression.