CheX in the three-phosphatase system of bacterial chemotaxis

Bacterial chemotaxis involves the regulation of motility by a modified two-component signal transduction system. In Escherichia coli, CheZ is the phosphatase of the response regulator CheY but many other bacteria, including Bacillus subtilis, use members of the CheC-FliY-CheX family for this purpose. While Bacillus subtilis has only CheC and FliY, many systems also have CheX. The effect of this three-phosphatase system on chemotaxis has not been studied previously. CheX was shown to be a stronger CheY-P phosphatase than either CheC or FliY. In Bacillus subtilis, a cheC mutant strain was nearly complemented by heterologous cheX expression. CheX was shown to overcome the DeltacheC adaptational defect but also generally lowered the counterclockwise flagellar rotational bias. The effect on rotational bias suggests that CheX reduced the overall levels of CheY-P in the cell and did not truly replicate the adaptational effects of CheC. Thus, CheX is not functionally redundant to CheC and, as outlined in the discussion, may be more analogous to CheZ.     

CheC and CheD interact to regulate methylation of Bacillus subtilis methyl-accepting chemotaxis proteins

In this study, we have demonstrated that two unique proteins in Bacillus subtilis chemotaxis, CheC and CheD, interact. We have shown this interaction both by using the yeast two-hybrid system and by precipitation of in vitro translated products using glutathione-S-transferase fusions and glutathione agarose beads. We have also shown that CheC inhibits B. subtilis CheR-mediated methylation of B. subtilis methyl-accepting chemotaxis proteins (MCPs) but not of Escherichia coli MCPs. It was previously reported that cheC mutants tend to swim smoothly and do not adapt to addition of attractant; cheD mutants have very poorly methylated MCPs and are very tumbiy, similar to cheA mutants. We hypothesize that CheC exerts its effect on MCP methylation in B. subtilis by controlling the binding of CheD to the MCPs. In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA. The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis in B. subtilis, whereby high levels of CheY-P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY-P.     

CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis

Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.     

The HtrA protease of Borrelia burgdorferi degrades outer membrane protein BmpD and chemotaxis phosphatase CheX

Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane‐bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B‐cell antigen. In co‐immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.     

Structure and function of HtrA family proteins, the key players in protein quality control

High temperature requirement A (HtrA) and its homologues constitute the HtrA family proteins, a group of heat shock-induced serine proteases. Bacterial HtrA proteins perform crucial functions with regard to protein quality control in the periplasmic space, functioning as both molecular chaperones and proteases. In contrast to other bacterial quality control proteins, including ClpXP, ClpAP, and HslUV, HtrA proteins contain no regulatory components or ATP binding domains. Thus, they are commonly referred to as ATP-independent chaperone-proteases. Whereas the function of ATP-dependent chaperone-proteases is regulated by ATP hydrolysis, HtrA exhibits a PDZ domain and a temperature-dependent switch mechanism, which effects the change in its function from molecular chaperone to protease. This mechanism is also related to substrate recognition and the fine control of its function. Structural and biochemical analyses of the three HtrA proteins, DegP, DegQ, and DegS, have provided us with clues as to the functional regulation of HtrA proteins, as well as their roles in protein quality control at atomic scales. The objective of this brief review is to discuss some of the recent studies which have been conducted regarding the structure and function of these HtrA proteins, and to compare their roles in the context of protein quality control.     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.     

The diverse CheC-type phosphatases: chemotaxis and beyond

A new class of protein phosphatases has emerged in the study of bacterial/archaeal chemotaxis, the CheC-type phosphatases. These proteins are distinct and unrelated to the well-known CheY-P phosphatase CheZ, though they have convergently evolved to dephosphorylate the same target. The family contains a common consensus sequence D/S-X(3)-E-X(2)-N-X(22)-P that defines the phosphatase active site, of which there are often two per protein. Three distinct subgroups make up the family: CheC, FliY and CheX. Further, the CheC subgroup can be divided into three classes. Bacillus subtilis CheC typifies the first class and might function as a regulator of CheD. Class II CheCs likely function as phosphatases in systems other than chemotaxis. Class III CheCs are found in the archaeal class Halobacteria and might function as class I CheCs. FliY is the main phosphatase in the B. subtilis chemotaxis system. CheX is quite divergent from the rest of the family, forms a dimer and some may function outside chemotaxis. A model for the evolution of the family is discussed.     

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.     

Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family

Reversible phosphorylation of cytoskeletal and cytoskeleton-associated proteins is a significant element of endothelial barrier function regulation. Therefore, understanding the mechanisms of phosphorylation/dephosphorylation of endothelial cell cytoskeletal proteins is vital to the treatment of severe lung disorders such as high permeability pulmonary edema. In vivo, there is a controlled balance between the activities of protein kinases and phosphatases. Due to various external or internal signals, this balance may be shifted. The actual balances at a given time alter the phosphorylation level of certain proteins with appropriate physiological consequences. The latest information about the structure and regulation of different types of Ser/Thr protein phosphatases participating in the regulation of endothelial cytoskeletal organization and barrier function will be reviewed here.     

Co-expression of signaling proteins improves robustness of the bacterial chemotaxis pathway

Biological systems are exposed to various perturbations that affect performance of the cellular networks, with stochastic variation in protein levels, or gene expression noise, being one of the major sources of intracellular perturbations. We recently used Escherichia coli chemotaxis as a model to analyze robustness against such noise and demonstrated theoretically and experimentally that a steady-state output of the pathway is robust against concerted variation in the levels of all chemotaxis proteins. However, our model predicted that the pathway topology does not confer much robustness against an uncorrelated variation in the protein levels. To test whether additional robustness features might be missing from our model, we compare here its predictions with an experimentally determined chemotactic performance under varying levels of individual proteins. Our data show that the pathway is indeed even more robust than predicted to two types of perturbations-the variation in the levels of the adaptation enzymes and a correlated expression of CheY and CheZ. Although the design features that are responsible for this higher robustness still remain to be understood, our results stress the importance of a robust design of both native and synthetic signaling networks.     

拟南芥SRO蛋白家族的结构及功能分析

许多生物及非生物胁迫都会引起植物的氧化胁迫, 参与植物氧化胁迫反应组分的鉴定备受人们的关注。拟南芥SRO家族成员包括AtRCD1、AtSRO1、AtSRO5等, 调节植物对氧化胁迫的反应。AtSROs参与植物正常的生长发育, 同时在植物应对干旱、盐、重金属等胁迫反应中扮演重要角色。AtSROs存在保守的PARP、RST等特殊功能区, 推测其可能具备蛋白的转录、调节、修饰等功能。文章就拟南芥SRO家族成员的基本状况, 在植物生长发育及应对非生物胁迫反应中的作用进行概述, 为进一步研究AtSROs的生物学功能提供理论基础。     

Purification and characterization of the CheZ protein of bacterial chemotaxis.

The cheZ gene is the most distal of five genes that comprise the Meche operon of the Salmonella typhimurium chemotaxis system. We have determined the sequence of the cheZ gene along with an 800-nucleotide flanking region at its 3' end. The flanking sequence contains an open reading frame that probably corresponds to the 5' end of flaM. The cheZ coding sequence predicts an extremely acidic, hydrophilic protein with a molecular weight of 23,900. We have purified and characterized this protein. N-terminal analysis of pure CheZ yields an amino acid sequence identical to that predicted by the nucleotide sequence except that the amino-terminal methionine residue is modified by N methylation. The purified CheZ protein exhibits a native molecular weight of 115,000, but in cell extracts the majority of CheZ exists as a much larger aggregate (Mr greater than 500,000). Under these conditions, CheZ appears to be a homopolymer composed of at least 20 monomeric subunits.     

Growth arrest–specific 2 protein family: Structure and function

Members of the growth arrest–specific 2 (GAS2) protein family consist of a putative actin‐binding (CH) domain and a microtubule‐binding (GAR) domain and are considered miniversions of spectraplakins. There are four members in the GAS2 family, viz. GAS2, GAS2L1, GAS2L2 and GAS2L3. Although GAS2 is defined as a family of growth arrest–specific proteins, the significant differences in the expression patterns, interaction characteristics and biological issues or diseases among the different GAS2 family members have not been systemically reviewed to date. Therefore, we summarized the available evidence on the structures and functions of GAS2 family members. This review facilitates a comprehensive molecular understanding of the involvement of the GAS2 family members in an array of biological processes, including cytoskeleton reorganization, cell cycle, apoptosis and cancer development.     

The dynamics of protein phosphorylation in bacterial chemotaxis

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.     

Structure, function and dynamics in the mur family of bacterial cell wall ligases

For bacteria, the structural integrity of its cell wall is of utmost importance for survival, and to this end, a rigid scaffold called peptidoglycan, comprised of sugar molecules and peptides, is synthesized and located outside the cytoplasmic membrane of the cell. Disruption of this peptidoglycan layer has for many years been a prime target for effective antibiotics, namely the penicillins and cephalosporins. Because this rigid layer is synthesized by a multi-step pathway numerous additional targets also exist that have no counterpart in the animal cell. Central to this pathway are four similar ligase enzymes, which add peptide groups to the sugar molecules, and interrupting these steps would ultimately prove fatal to the bacterial cell. The mechanisms of these ligases are well understood and the structures of all four of these ligases are now known. A detailed comparison of these four enzymes shows that considerable conformational changes are possible and that these changes, along with the recruitment of two different N-terminal binding domains, allows these enzymes to bind a substrate which at one end is identical and at the other has the growing polypeptide tail. Some insights into the structure-function relationships in these enzymes is presented.