Protein tyrosine phosphatase function: the substrate perspective

It is now well established that the members of the PTP (protein tyrosine phosphatase) superfamily play critical roles in fundamental biological processes. Although there has been much progress in defining the function of PTPs, the task of identifying substrates for these enzymes still presents a challenge. Many PTPs have yet to have their physiological substrates identified. The focus of this review will be on the current state of knowledge of PTP substrates and the approaches used to identify them. We propose experimental criteria that should be satisfied in order to rigorously assign PTP substrates as bona fide. Finally, the progress that has been made in defining the biological roles of PTPs through the identification of their substrates will be discussed.     

Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.     

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

Substrate Specificity of R3 Receptor-like Protein-tyrosine Phosphatase Subfamily toward Receptor Protein-tyrosine Kinases

Receptor-like protein-tyrosine phosphatases (RPTPs) are involved in various aspects of cellular functions, such as proliferation, differentiation, survival, migration, and metabolism. A small number of RPTPs have been reported to regulate activities of some cellular proteins including receptor protein-tyrosine kinases (RPTKs). However, our understanding about the roles of individual RPTPs in the regulation of RPTKs is still limited. The R3 RPTP subfamily reportedly plays pivotal roles in the development of several tissues including the vascular and nervous systems. Here, we examined enzyme-substrate relationships between the four R3 RPTP subfamily members and 21 RPTK members selected from 14 RPTK subfamilies by using a mammalian two-hybrid system with substrate-trapping RPTP mutants. Among the 84 RPTP-RPTK combinations conceivable, we detected 30 positive interactions: 25 of the enzyme-substrate relationships were novel. We randomly chose several RPTKs assumed to be substrates for R3 RPTPs, and validated the results of this screen by in vitro dephosphorylation assays, and by cell-based assays involving overexpression and knock-down experiments. Because their functional relationships were verified without exception, it is probable that the RPTKs identified as potential substrates are actually physiological substrates for the R3 RPTPs. Interestingly, some RPTKs were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily.     

Macromolecular substrates for the ICE-like proteases during apoptosis

The interleukin-1β-converting enzyme (ICE) family of proteases is an important component of the mechanism of the apoptotic process, but the physiologic roles of the different homologs during apoptosis remain unclear. Significant information about the roles of proteolysis in apoptosis will be gained through identification of the distal substrates through which these proteases achieve their pro-apoptotic effects. Identification of these substrates therefore remains an important challenge. A subset of autoantibodies from patients with systemic lupus erythematosus (SLE) recognize molecules that are specifically cleaved early during apoptosis. Several of the identified autoantigens are nuclear proteins (PARP, U1-70 kDa, and DNA-PK_CS) that are substrates for CPP32 in vitro and in apoptotic cells. Of note, these substrates are catalytic proteins involved in homeostatic pathways, suggesting that abolition of homeostasis is one fundamental feature ensuring the rapid irreversibility of the apoptotic process. Identification of the other substrates for this protease family will provide the tools to assess the roles of the different proteases in apoptotic death. J. Cell. Biochem. 64:50–54. © 1997 Wiley-Liss, Inc.     

Receptor tyrosine kinase substrates: src homology domains and signal transduction.

Among the intracellular milieu of proteins are molecules with defined biochemical functions that serve as substrates for ligand-activated tyrosine kinase receptors. It seems likely that some of these substrate molecules are elements of a critical signaling pathway used by growth factors to control cell proliferation and subverted by oncogenes to deregulate this process. Although the process of cell growth and division is relatively slow compared with other hormonally regulated responses, homeostasis in a human being requires approximately 20 x 10(6) cell divisions per second for the renewal of various cell populations. This review summarizes the present understanding of tyrosine kinase substrates that seem likely to have key roles in the signal transduction pathway that regulates cell proliferation. This includes structural features of these molecules, the influence of tyrosine phosphorylation on their functions, the biological roles of these proteins, and the capacity of these substrates to associate with activated receptor tyrosine kinases.     

The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.     

Characterization of O-methyltransferase ScOMT1 cloned from Streptomyces coelicolor A3(2)

The roles of O-methyltransferases (OMTs) in microorganisms are not well understood, and are suggested to increase antimicrobial activity. Studies on OMTs cloned from microorganisms may help elucidate their roles. Streptomyces coelicolor A3(2) produces many useful natural antibiotics such as actinorhodin. Based on sequence information from S. coelicolor A3(2) genome, it was possible to clone several methyltransferases. An OMT cloned from Streptomyces coelicolor A3(2), ScOMT1 was characterized by in vivo and in vitro assays. Of 23 compounds tested, 13 were found to serve as its substrates. Of the 13 substrates, the methylated positions of 7 compounds were determined by HPLC, NMR, and MS analyses. This OMT favored ortho-dihydroxyflavones. Among the compounds tested here, the best substrate is 6,7-dihydroxyflavone.     

Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics

Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.     

Targeting exported substrates to the Yersinia TTSS: different functions for different signals?

Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.     

Plant terpenes and lignin as natural cosubstrates in biodegradation of polyclorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs)

The objective of this minireview is to examine how cometabolic biodegradation of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) might be affected by plant terpenes and lignins as natural substrates abundant in nature. The topics covered, hence, are environmental significance of PCBs and PAHs, nature and distribution of plant terpenes and lignin, structural and metaoblic similarities of the natural compounds to PCBs and PAHs, and possible roles of the natural substrates in inducing the biodegradative pathways of PCBs and PAHs.     

Quantitative proteomic identification of the BRCA1 ubiquitination substrates

Mutation of the BRCA1 tumor suppressor gene predisposes women to hereditary breast and ovarian cancers. BRCA1 forms a heterodimer with BARD1. The BRCA1/BARD1 heterodimer has ubiquitin ligase activity, considered to play crucial roles in tumor suppression and DNA damage response. Nevertheless, relevant BRCA1 substrates are poorly defined. We have developed a new approach to systematically identify the substrates of ubiquitin ligases by identifying proteins that display an enhanced incorporation of His-tagged ubiquitin upon ligase coexpression; using this method, we identified several candidate substrates for BRCA1. These include scaffold attachment factor B2 (SAFB2) and Tel2 as well as BARD1. BRCA1 was found to enhance SAFB protein expression and induce Tel2 nuclear translocation. Identification of the ubiquitination substrates has been a major obstacle to understanding the functions of ubiquitin ligases. The quantitative proteomics approach we devised for the identification of BRCA1 substrates will facilitate the identification of ubiquitin ligase-substrate pairs.     

新型工业酶制剂的进步对生物化学品工业生产过程的影响

工业酶制剂对化工和生物化工产品生产的影响有两方面,一是直接催化生产,另一方面是辅助发酵菌进行生产。以下简单回顾了酶制剂在工业领域的应用现状,注重讨论酶制剂在淀粉糖等方面的直接催化应用和在酒精发酵生产等方面的辅助作用,分析新型酶制剂对生产过程的影响及带来的益处,促进行业的可持续发展。     

The role of A Disintegrin and Metalloproteinase (ADAM)-10 in T helper cell biology

A Disintegrin and Metalloproteinases (ADAM)-10 is a member of a family of membrane-anchored proteinases that regulate a broad range of cellular functions with central roles within the immune system. This has spurred the interest to modulate ADAM activity therapeutically in immunological diseases. CD4 T helper (Th) cells are the key regulators of adaptive immune responses. Their development and function is strongly dependent on Notch, a key ADAM-10 substrate. However, Th cells rely on a variety of additional ADAM-10 substrates regulating their functional activity at multiple levels. The complexity of both, the ADAM substrate expression as well as the functional consequences of ADAM-mediated cleavage of the various substrates complicates the analysis of cell type specific effects.Here we provide an overview on the major ADAM-10 substrates relevant for CD4 T cell biology and discuss the potential effects of ADAM-mediated cleavage exemplified for a selection of important substrates.     

Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart

A method to study the export of citric acid cycle intermediates from rat liver mitochondria supplied with various individual substrates or combinations of substrates was designed to focus on the role of mitochondria in anaplerosis and cataplerosis. Under most conditions malate, citrate, and aspartate were exported in far higher amounts than isocitrate and alpha-ketoglutarate. In the presence of pyruvate alone or pyruvate in combination with most other substrates, citrate export equaled or was only slightly less than malate export. This contrasts with pancreatic islet mitochondria where citrate export is unaffected by many substrates. Malate and succinate potentiated pyruvate-induced citrate export and succinate caused massive malate export from liver mitochondria. Heart mitochondria, which possess very little or no pyruvate carboxylase, unlike liver and pancreatic islet mitochondria, did not produce malate from pyruvate. Heart mitochondria produced malate, but not citrate, from succinate. The results indicate that liver mitochondria export a larger number of metabolites from a wider range of substrates than do islet or heart mitochondria. This may reflect the multiple roles of the liver in body metabolism versus the specialized roles of the islet cell and heart.     

Multilevel structural characteristics for the natural substrate proteins of bacterial small heat shock proteins

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones that prevent the aggregation of various non‐native proteins and play crucial roles for protein quality control in cells. It is poorly understood what natural substrate proteins, with respect to structural characteristics, are preferentially bound by sHSPs in cells. Here we compared the structural characteristics for the natural substrate proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with the respective bacterial proteome at multiple levels, mainly by using bioinformatics analysis. Data indicate that both IbpB and Hsp20.2 preferentially bind to substrates of high molecular weight or moderate acidity. Surprisingly, their substrates contain abundant charged residues but not abundant hydrophobic residues, thus strongly indicating that ionic interactions other than hydrophobic interactions also play crucial roles for the substrate recognition and binding of sHSPs. Further, secondary structure prediction analysis indicates that the substrates of low percentage of β‐sheets or coils but high percentage of α‐helices are un‐favored by both IbpB and Hsp20.2. In addition, IbpB preferentially interacts with multi‐domain proteins but unfavorably with α + β proteins as revealed by SCOP analysis. Together, our data suggest that bacterial sHSPs, though having broad substrate spectrums, selectively bind to substrates of certain structural features. These structural characteristic elements may substantially participate in the sHSP–substrate interaction and/or increase the aggregation tendency of the substrates, thus making the substrates more preferentially bound by sHSPs.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Roles of the ubiquitin-proteosome system in neurogenesis

The ubiquitin-proteosome system (UPS) is a non-lysosomal proteolysis system involved in the degradation of irrelevant/misfolded intracellular proteins. The protein substrates of this system are tagged by ubiquitin in sequential reactions that target them for proteasome-dependent destruction. In the developing central nervous system, ubiquitin-mediated proteolysis has recently emerged as an important player in the regulation of neural progenitor proliferation, cell specification, neuronal differentiation, maturation, and migration. E3 ubiquitin ligases are crucial components in the UPS because they provide the specificity that determines which substrates are targeted for ubiquitin-dependent proteolysis. In this review, we discuss the molecular mechanisms of the UPS, focusing primarily on the roles of E3 ligases and their substrates in sequential steps of neurogenesis.