The GGA proteins: key players in protein sorting at the trans-Golgi network

The GGA (Golgi-localized, gamma-ear containing, ADP-ribosylation factor binding) family of multidomain coat proteins was first described in the year 2000. They are now known to occupy a central position in the trafficking of the mannose 6-phosphate receptors and other cargo molecules from the trans-Golgi network to the endosome/lysosome system. This review covers the recent structural and cell biological studies that have provided mechanistic insights into the function of the GGAs in mannose 6-phosphate receptor trafficking.     

Structure, function, and evolution of the PsbP protein family in higher plants

The PsbP is a thylakoid lumenal subunit of photosystem II (PSII), which has developed specifically in higher plants and green algae. In higher plants, the molecular function of PsbP has been intensively investigated by release-reconstitution experiments in vitro. Recently, solution of a high-resolution structure of PsbP has enabled investigation of structure-function relationships, and efficient gene-silencing techniques have demonstrated the crucial role of PsbP in PSII activity in vivo. Furthermore, genomic and proteomic studies have shown that PsbP belongs to the divergent PsbP protein family, which consists of about 10 members in model plants such as Arabidopsis and rice. Characterization of the molecular function of PsbP homologs using Arabidopsis mutants suggests that each plays a distinct and important function in maintaining photosynthetic electron transfer. In this review, recent findings regarding the molecular functions of PsbP and other PsbP homologs in higher plants are summarized, and the molecular evolution of these proteins is discussed.     

Argonaute proteins: key players in RNA silencing

During the past decade, small non-coding RNAs have rapidly emerged as important contributors to gene regulation. To carry out their biological functions, these small RNAs require a unique class of proteins called Argonautes. The discovery and our comprehension of this highly conserved protein family is closely linked to the study of RNA-based gene silencing mechanisms. With their functional domains, Argonaute proteins can bind small non-coding RNAs and control protein synthesis, affect messenger RNA stability and even participate in the production of a new class of small RNAs, Piwi-interacting RNAs.     

Prion proteins meet protein quality control

Recent work on transmissible spongiform encephalopathies (TSEs) suggests a role for protein quality-control mechanisms in both prion protein aggregation and pathogenesis. Cytosolic accumulation of prion protein seems to be neurotoxic and might occur when proteasome function is compromised and quality control is overwhelmed. These findings are discussed in the light of other studies linking proteasome inhibition and neurodegeneration.     

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 an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX

In bacterial chemotaxis, phosphorylated CheY levels control the sense of flagella rotation and thereby determine swimming behavior. In E. coli, CheY dephosphorylation by CheZ extinguishes the switching signal. But, instead of CheZ, many chemotactic bacteria contain CheC, CheD, and/or CheX. The crystal structures of T. maritima CheC and CheX reveal a common fold unlike that of any other known protein. Unlike CheC, CheX dimerizes via a continuous beta sheet between subunits. T. maritima CheC, as well as CheX, dephosphorylate CheY, although CheC requires binding of CheD to achieve the activity of CheX. Structural analyses identified one conserved active site in CheX and two in CheC; mutations therein reduce CheY-phosphatase activity, but only mutants of two invariant asparagine residues are completely inactive even in the presence of CheD. Our structures indicate that the flagellar switch components FliY and FliM resemble CheC more closely than CheX, but attribute phosphatase activity only to FliY.     

Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria.

The protein sequences of seven members of the superoxide dismutase (SOD) family from halophilic archaebacteria have been aligned and compared with each other and with the homologous Mn and Fe SOD sequences from eubacteria and the methanogenic archaebacterium Methanobacterium thermoautotrophicum. Of 199 common residues in the SOD proteins from halophilic archaebacteria, 125 are conserved in all seven sequences, and 64 of these are encoded by single unique triplets. The 74 remaining positions exhibit a high degree of variability, and for almost half of these, the encoding triplets are connected by at least two nonsynonymous nucleotide substitutions. The majority of nucleotide substitutions within the seven genes are nonsynonymous and result in amino acid replacement in the respective protein; silent third-codon-position (synonymous) substitutions are unexpectedly rare. Halophilic SODs contain 30 specific residues that are not found at the corresponding positions of the methanogenic or eubacterial SOD proteins. Seven of these are replacements of highly conserved amino acids in eubacterial SODs that are believed to play an important role in the three-dimensional structure of the protein. Residues implicated in formation of the active site, catalysis, and metal ion binding are conserved in all Mn and Fe SODs. Molecular phylogenies based on parsimony and neighbor-joining methods coherently group the halophile sequences but surprisingly fail to distinguish between the Mn SOD of Escherichia coli and the Fe SOD of M. thermoautotrophicum as the outgroup. These comparisons indicate that as a group, the SODs of halophilic archaebacteria have many unique and characteristic features. At the same time, the patterns of nucleotide substitution and amino acid replacement indicate that these genes and the proteins that they encode continue to be subject to strong and changing selection. This selection may be related to the presence of oxygen radicals and the inter- and intracellular composition and concentration of metal cations.     

Structure and function of the guanylate kinase-like domain of the MAGUK family scaffold proteins

Membrane associated guanylate kinases (MAGUKs) are a family of scaffold proteins that play essential roles in organ development, cell-cell communication, cell polarity establishment and maintenance, and cellular signal transduction. Every member of the MAGUK family contains a guanylate kinase-like (GK) domain, which has evolved from the enzyme catalyzing GMP to GDP conversion to become a protein-protein interaction module with no enzymatic activity.Mutations of MAGUKs are linked to a number of human diseases, including autism and hereditary deafness. In this review, we summarize the structural basis governing cellular function of various members of the MAGUKs. In particular, we focus on recent discoveries of MAGUK GKs as specific phospho-protein interaction modules, and discuss functional implications and connections to human diseases of such regulated MAGUK GK/target interactions.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

Dead-box proteins: a family affair--active and passive players in RNP-remodeling

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.     

Mitogen-activated protein kinases as key players in osmotic stress signaling

BackgroundOsmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.Scope of reviewThe present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.Major conclusionsMAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.General significanceMAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.     

Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis

Staphylococcus aureus encodes two HtrA-like serine surface proteases, called HtrA1 and HtrA2. The roles of these HtrA homologs were distinguished by expression studies in a heterologous host, Lactococcus lactis, whose single extracellular protease, HtrA(Ll), was absent. HtrA(Ll) is involved in stress resistance, and processing and/or degradation of extracellular proteins. Controlled expression of staphylococcal htrA1 and htrA2 was achieved in L. lactis strain NZ9000 DeltahtrA, as confirmed with anti-HtrA1 and anti-HtrA2 specific antibodies. HtrA1 fully restored thermo-resistance to the htrA-defective L. lactis strain, despite a poor capacity to degrade abnormal or truncated proteins. We therefore propose that activities of HtrA1 other than proteolysis may be sufficient for high-temperature growth complementation. HtrA2 is 36% identical to HtrA(Ll), and was highly expressed in L. lactis; nevertheless, it displayed nearly no detectable activities. The poor proteolytic activities of staphylococcal HtrA proteins in L. lactis may reflect a requirement for S. aureus-specific co-factors.     

The HtrA family of proteases: implications for protein composition and cell fate

Cells precisely monitor the concentration and functionality of each protein for optimal performance. Protein quality control involves molecular chaperones, folding catalysts, and proteases that are often heat shock proteins. One quality control factor is HtrA, one of a new class of oligomeric serine proteases. The defining feature of the HtrA family is the combination of a catalytic domain with at least one C-terminal PDZ domain. Here, we discuss the properties and roles of this ATP-independent protease chaperone system in protein metabolism and cell fate.     

Snake venom metalloproteinases: Structure, function and relevance to the mammalian ADAM/ADAMTS family proteins

Metalloproteinases are among the most abundant toxins in many Viperidae venoms. Snake venom metalloproteinases (SVMPs) are the primary factors responsible for hemorrhage and may also interfere with the hemostatic system, thus facilitating loss of blood from the vasculature of the prey. SVMPs are phylogenetically most closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif) family of proteins and, together with them, constitute the M12B clan of metalloendopeptidases. Large SVMPs, referred to as the P-III class of SVMPs, have a modular architecture with multiple non-catalytic domains. The P-III SVMPs are characterized by higher hemorrhagic and more diverse biological activities than the P-I class of SVMPs, which only have a catalytic domain. Recent crystallographic studies of P-III SVMPs and their mammalian counterparts shed new light on structure-function properties of this class of enzymes. The present review will highlight these structures, particularly the non-catalytic ancillary domains of P-III SVMPs and ADAMs that may target the enzymes to specific substrates. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Structure, function and evolution of multidomain proteins

Proteins are composed of evolutionary units called domains; the majority of proteins consist of at least two domains. These domains and nature of their interactions determine the function of the protein. The roles that combinations of domains play in the formation of the protein repertoire have been found by analysis of domain assignments to genome sequences. Additional findings on the geometry of domains have been gained from examination of three-dimensional protein structures. Future work will require a domain-centric functional classification scheme and efforts to determine structures of domain combinations.