Overview: development in bacteria: spore formation in Bacillus subtilis

Like eukaryotes, bacteria possess complex developmental programs that drive environmental adaptation and morphological differentiation. In some species, these morphological changes are quite elaborate and result in major changes in cell appearance, including the formation of ornate appendages. The ease with which some bacteria can be manipulated makes them highly attractive model systems for developmental analysis. In this set of reviews, we tackle the best studied of these systems, spore formation in Bacillus subtilis. Construction of a spore initiates in response to starvation, takes each cell about 8 h and is directed by a tightly controlled genetic program. First, the cell creates an internal protoplast with its own copy of the chromosome. Over the next several hours, development continues as proteins synthesized within the protoplast as well as in the surrounding cell cytoplasm coalesce into the various complex structures that comprise the spore. The resulting cell is metabolically dormant and as close to indestructible as any cell found on earth. Nonetheless, the spore retains the ability to revive almost immediately when nutrient returns to the environment. Here, we review the genetic control of spore formation, the structure and assembly of several major spore components, the process of germination, and the environmental and disease implications of spores. As these reviews document, spore formation in B. subtilis has been among the most productive systems for understanding both the broad themes and the molecular basis of development. Not only does this system continue to add to our understanding of these questions, but it provides a particularly powerful means to address the cell biological dimension of development.     

A role for the hinge/ear domain of the β chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the β2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated β2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the β chains indeed provide an important interaction between the AP complexes and clathrin. Received 7 April 1997; received after revision 22 May 1997; accepted 28 May 1997     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Structure and assembly of the 20S proteasome

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain α-type subunits, the inner ones comprise β-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the α-type and β-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the β-type subunits are processed. The prosequence of the β-type subunits is essential for the assembly process and prevents protease activity of immature proteasomes.     

Structure, function and evolution of antifreeze proteins

Antifreeze proteins bind to ice crystals and modify their growth. These proteins show great diversity in structure, and they have been found in a variety of organisms. The ice-binding mechanisms of antifreeze proteins are not completely understood. Recent findings on the evolution of antifreeze proteins and on their structures and mechanisms of action have provided new understanding of these proteins in different contexts. The purpose of this review is to present the developments in contrasting research areas and unite them in order to gain further insight into the structure and function of the antifreeze proteins. Received 2 September 1998; received after revision 21 October 1998; accepted 2 November 1998     

基于Pro／TOOLKIT的截割头截齿及齿座虚拟装配

通过建立单个截齿及齿座的定位模型,应用PROE的二次开发工具包Pro／TOOLKIT、VC＋＋和数据库技术,开发出掘进机截割头截齿及齿座的虚拟装配程序。该程序只需输入所有截齿定位模型的参数数据,即可得到整个截割头截齿与齿座的装配体。由此装配体能确定各个齿座的安装平面。进而得到满足设计要求的截割头头体,实现整个截割头的虚拟装配。为质量高、性能好的掘进机截割头的研发提供了一种高效、快捷的途径。     

sHsps and their role in the chaperone network

Small Hsps (sHsps) encompass a widespread but diverse class of proteins. These low molecular mass proteins (15—42 kDa) form dynamic oligomeric structures ranging from 9 to 50 subunits. sHsps display chaperone function in vitro, and in addition they have been suggested to be involved in the inhibition of apoptosis, organisation of the cytoskeleton and establishing the refractive properties of the eye lens in the case of α-crystallin. How these different functions can be explained by a common mechanism is unclear at present. However, as most of the observed phenomena involve nonnative protein, the repeatedly reported chaperone properties of sHsps seem to be of key importance for understanding their function. In contrast to other chaperone families, sHsps bind several nonnative proteins per oligomeric complex, thus representing the most efficient chaperone family in terms of the quantity of substrate binding. In some cases, the release of substrate proteins from the sHsp complex is achieved in cooperation with Hsp70 in an ATP-dependent reaction, suggesting that the role of sHsps in the network of chaperones is to create a reservoir of nonnative refoldable protein.     

NMR structures of the C-terminal end of human complement serine protease C1s

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular α-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue α-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672. Received 19 November 1997; accepted 24 November 1997     

Noncollagenous, nonproteoglycan macromolecules of cartilage

Extracellular matrix comprises approximately 90% of cartilage, with collagens and proteoglycans making up the bulk of the tissue. In recent years, several abundant cartilage proteins that are neither collagens nor proteoglycans have been characterized in detail. The putative roles of these proteins range from involvement in matrix organization or matrix-cell signaling (PRELP, chondroadherin, cartilage oligomeric protein and cartilage matrix protein) through to molecules that are likely to be involved with modulation of the chondrocyte phenotype (CD-RAP, CDMPs, chondromodulin and pleiotrophin). Other molecules, such as the cartilage-derived C-type lectin and cartilage intermediate layer protein have no role as yet. Due to the difficulties associated with experimentally manipulating a tissue that is 90% extracellular matrix in a manner that can be readily transferred to the whole organism, many of these molecules have been focused on by a surprisingly small number of researchers. This review focuses on newly discovered proteins and glycoproteins in cartilage, with a bias towards those that have structural roles or that are unique to cartilage. Received 7 January 1999; accepted 11 March 1999     

Diversity of structures and properties among catalases

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.Received 2 June 2003; received after revision 24 June 2003; accepted 1 July 2003     

Molecular modeling of mammalian cytochrome P450s

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure, models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s. Received 28 September 1999; received after revision 25 November 1999; accepted 31 December 1999     

The surface glycopeptidolipids of mycobacteria: structures and biological properties

One of the most important opportunistic pathogens associated with acquired immunodeficiency syndrome (AIDS) is the M. avium complex. M. avium infections are found in up to 70% of individuals in advanced stages of AIDS. It is apparent that M. avium can replicate in host macrophages and persist for long periods. This group of mycobacteria are distinguished by the presence of unique, highly antigenic, surface-located lipids known as the glycopeptidolipids (GPLs). The GPLs are the chemical basis of the 31 distinct serovars of the M. avium complex, and have also been identified in some other species. The M. avium lipids are immunosuppressive and can induce a variety of cytokines that affect general host responses. Despite extensive chemical characterization of the structures of these GPLs, much work is needed to elucidate the molecular mechanism involved in this complex glycosylation pathway and its genetic basis. The challenges for the future lie in explaining the roles of these copious products in the intracellular life and infectivity of mycobacteria. The intention of our review is to offer a concise account of the structures of the M. avium lipids, their putative roles in the host responses, bacterial physiology and pathogenesis, particularly in immunocompromised patients such as those infected with human immunodeficiency virus (HIV). Advances in chemical synthesis of the various haptenic oligosaccharides are also given to demonstrate how these have helped to define the immunogenic determinants. We believe that future research should involve the creation of conditional mutants defective in these lipids for both functional and biosynthesis studies which will complement biological assays using chemically defined or modified neoglycoconjugates. Received 7 May 2001; received after revision 28 June 2001; accepted 28 June 2001     

T-cell signal transduction and the role of protein kinase C

The T lymphocyte has a vital part to play in maintaining the host response to bacterial and viral infection and also appears to play a key pathological role in autoimmune diseases such as rheumatoid arthritis. In this review, we summarize the signalling pathways which trigger antigen-driven T-cell proliferation and examine the evidence which suggests that protein kinase C (PKC) is fundamental to this process. Finally, we discuss the therapeutic potential that PKC inhibitors may have in the treatment of autoimmune disease. Received 31 March 1998; received after revision 19 May 1998; accepted 19 May 1998     

Synthetic genes for the elucidation of glycosylation codes for arabinogalactan-proteins and other hydroxyproline-rich glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of the growing plant cell wall, accounting for as much as 10-20% of the dry weight. HRGPs are implicated in all aspects of plant growth and development, including responses to stress. The HRGP superfamily contains three major groups which represent a continuum of peptide periodicity and hydroxyproline-O-glycosylation. These groups range from the highly periodic and lightly arabinosylated repetitive proline-rich proteins (PRPs), through the crosslinked extensins which are periodic and highly arabinosylated, to the arabinogalactan-proteins (AGPs) which are the most highly glycosylated and least periodic. The repetitive units are small, often only four- to six-residue-glycosylated modules viewed hypothetically as functional motifs, or glycomodules. The Hyp contiguity hypothesis predicts that Hyp arabinosylation increases with Hyp contiguity and that clustered noncontiguous Hyp residues are sites of arabinogalactan polysaccharide addition in the AGPs and gums. Recent results involving glycosylation site mapping of endogenous HRGPs and HRGP design using synthetic genes have corroborated the hypothesis. The uses of synthetic genes in HRGP glycosylation site mapping and structural/functional analysis are also discussed.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

The structure and function of Escherichia coli penicillin-binding protein 3

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.     

Phosphodiesterase: overview of protein structures, potential therapeutic applications and recent progress in drug development

Phosphodiesterases (PDEs) are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Because of their great market potential and therapeutic importance, PDE inhibitors became recognized as important therapeutic agents in the treatment of various diseases. Currently, there are seven PDE inhibitors on the market, and the pharmacological and safety evaluations of many drug candidates are in progress. Three-dimensional (3D) structures of catalytic domains of PDE 1, -3, -4, -5 and -9 in the presence of their inhibitors are now available, and can be utilized for rational drug design. Recent advances in molecular pharmacology of PDE isoenzymes resulted in identification of new potential applications of PDE inhibitors in various therapeutic areas, including dementia, depression and schizophrenia. This review will describe the latest advances in PDE research on 3D structural studies, the potential of therapeutic applications and the development of drug candidates.Received 30 November 2004; received after revision 24 January 2005; accepted 5 February 2005     

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen

The life of aerobes is dependent on iron and oxygen for efficient bioenergetics. Due to potential risks associated with iron/oxygen chemistry, iron acquisition, concentration, storage, utilization, and efflux are tightly regulated in the cell. A central role in regulating iron/oxygen chemistry in animals is played by mRNA translation or turnover via the iron responsive element (IRE)/iron regulatory protein (IRP) system. The IRE family is composed of three-dimensional RNA structures located in 3′ or 5′ untranslated regions of mRNA. To date, there are 11 different IRE mRNAs in the family, regulated through translation initiation or mRNA stability. Iron or oxidant stimuli induce a set of graded responses related to mRNA-specific IRE substructures, indicated by differential responses to iron in vivo and binding IRPs in vitro. Molecular effects of phosphorylation, iron and oxygen remain to be added to the structural information of the IRE-RNA and IRP repressor in the regulatory complex. Received 21 April 2007; received after revision 13 July 2007; accepted 2 August 2007     

Protein misfolding and disease: the case of prion disorders

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies. Received 15 July 2002; received after revision 19 August 2002; accepted 23 August 2002 RID="*" ID="*"Corresponding author.     

Clostridial neurotoxins: structure-function led design of new therapeutics

The neurotoxins produced by various species of Clostridia are the causative agents of botulism and tetanus. The ability of the toxins, specifically those of the botulinum neurotoxin family, to disrupt neurotransmission has been exploited for use in several medical indications and now represents the therapeutic option of choice in a number of cases. Clostridial neurotoxins have been discovered to have a multi-domain structure that is shared between the various proteins of the family, and it has also been determined that each domain contributes a specific role to the holotoxin. The extensive use of recombinant expression approaches, along with solution of multiple crystallographic structures of individual domains, has enabled researchers to explore structurefunction relationships of the toxin domains more closely. These advances have facilitated a greater understanding of the potential use of individual domains for a wide variety of purposes, including the development of new therapeutics. Received 21 October 2005; received after revision 10 November 2005; accepted 16 November 2005