pHi regulatory ion transporters: an update on structure, regulation and cell function

Intracellular pH (pH_i) is a major regulator of various and critical cellular functions. A close regulation of pH_i is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H⁺ or equivalent H⁺ into and out of the cell. Besides pH_i, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters. Received 30 December 1998; received after revision 4 February 1999; accepted 9 February 1999     

The alpha-kinase family: an exceptional branch on the protein kinase tree

The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg²⁺ homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.     

The Janus kinase family of protein tyrosine kinases and their role in signaling

In the early 1990s, the search for protein kinases led to the discovery of a novel family of non-receptor tyrosine kinases, the Janus kinases or JAKs. These proteins were unusual because they contained two kinase homology domains and no other known signaling modules. It soon became clear that these were not ‘just another’ type of kinase. Their ability to complement mutant cells insensitive to interferons and to be activated by a variety of cytokines demonstrated their central signaling function. Now, as we approach the end of the decade, it is evident from biochemical studies to knockout mice that JAKs play non-redundant functions in development, differentiation, and host defense mechanisms. Here, recent progress is reviewed, with particular emphasis on structure-function studies aimed at revealing how this family of tyrosine kinases is regulated.     

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.     

The CorA family: Structure and function revisited

The CorA family is a group of ion transporters that mediate transport of divalent metal ions across biological membranes. Metal ions are essential elements in most cellular processes and hence the concentrations of ions in cells and organelles must be kept at appropriate levels. Impairment of these systems is implied in a number of pathological conditions. CorA proteins are abundant among the prokaryotic organisms but homologues are present in both human and yeast. The activity of CorA proteins has generally been associated with the transport of magnesium ions but the members of the CorA family can also transport other ions such as cobalt and nickel. The structure of the CorA from Thermotoga maritima, which also was the first structure of a divalent cation transporter determined, has opened the possibilities for understanding the mechanisms behind the ion transport and also corrected a number of assumptions that have been made in the past.     

HCN channels: Structure, cellular regulation and physiological function

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. HCN channels are unique among vertebrate voltage-gated ion channels, in that they have a reverse voltage-dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN channels are encoded by four genes (HCN1–4) and are widely expressed throughout the heart and the central nervous system. The current flowing through HCN channels, designated I_h or I_f, plays a key role in the control of cardiac and neuronal rhythmicity (“pacemaker current”). In addition, I_h contributes to several other neuronal processes, including determination of resting membrane potential, dendritic integration and synaptic transmission. In this review we give an overview on structure, function and regulation of HCN channels. Particular emphasis will be laid on the complex roles of these channels for neuronal function and cardiac rhythmicity. Received 22 August 2008; received after revision 22 September 2008; accepted 24 September 2008     

Role of serotonin in the hepato-gastroIntestinal tract: an old molecule for new perspectives

Beside its role as a neurotransmitter in the central nervous system, serotonin appears to be a central physiologic mediator of many gastrointestinal (GI) functions and a mediator of the brain-gut connection. By acting directly and via modulation of the enteric nervous system, serotonin has numerous effects on the GI tract. The main gut disturbances in which serotonin is involved are acute chemotherapy-induced nausea and vomiting, carcinoid syndrome and irritable bowel syndrome. Serotonin also has mitogenic properties. Platelet-derived serotonin is involved in liver regeneration after partial hepatectomy. In diseased liver, serotonin may play a crucial role in the progression of hepatic fibrosis and the pathogenesis of steatohepatitis. Better understanding of the role of the serotonin receptor subtypes and serotonin mechanisms of action in the liver and gut may open new therapeutic strategies in hepato-gastrointestinal diseases. Received 15 August 2007; received after revision 1 November 2007; accepted 5 November 2007     

Structure and function of Zika virus NS5 protein: perspectives for drug design

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure–function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed.     

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins. Received 30 September 2005; received after revision 5 November 2005; accepted 28 November 2005     

T helper 17 cells: discovery, function, and physiological trigger

In the few years since their discovery, T helper 17 cells (T_H17) have been shown to play an important role in host defense against infections, and in tissue inflammation during autoimmunity. T_H17 cells produce IL-17, IL-21, IL-10, and IL-22 cytokines, and thus have broad effects on a variety of tissues. Notably, the requirement for the immunosuppressive cytokine TGF-β along with the pro-inflammatory cytokine IL-6 for T_H17 differentiation supports the intimate relationship between the T_H17 subset and FOXP3⁺ regulatory T cells. Here, we discuss current knowledge on effector functions and differentiation of the T_H17 lineage. Furthermore, we now know of a physiological stimulus for T_H17 differentiation: innate immune recognition of cells undergoing apoptosis as a direct result of infection induces unique development of this subset. As our knowledge of T_H17 and T regulatory cells grows, we are building on a new framework for the understanding of effector T cell differentiation and the biology of CD4⁺ T cell adaptive immune responses.     

The Rh protein family: gene evolution, membrane biology, and disease association

The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor’s involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.     

Neuroactive steroids: State of the art and new perspectives

Neuroactive steroids include synthetic steroidal compounds and endogenous steroids, produced by endocrine glands (hormonal steroids) or the nervous tissue (neurosteroids), which regulate neural functions. These steroids bind to nuclear receptors or act through the activation of membrane-associated signaling pathways to modulate various important processes including the development of the nervous system, neural plasticity and the adaptive responses of neurons and glial cells under pathological conditions. Reviewed and updated in the present paper are the pleiotropic and protective abilities of neuroactive steroids. The fundamental evidence and knowledge gained constitute a profound background that offers interesting possibilities for developing effective strategies against several disorders of the nervous system. Received 3 September 2007; received after revision 24 October 2007; accepted 29 October 2007     

Microsatellite instability in gastric cancer: molecular bases,clinical perspectives,and new treatment approaches

Gastric cancer is one of the most aggressive malignancies, with limited treatment options in both locally advanced and metastatic setting, resulting in poor prognosis. Based on genomic characterization, stomach tumour has recently been described as a heterogeneous disease composed by different subtypes, each of them with peculiar molecular aspects and specific clinical behaviour. With an incidence of 22% among all western gastric tumour cases, stomach cancer with microsatellite instability was identified as one of these subgroups. Retrospective studies and limited prospective trials reported differences between gastric cancers with microsatellite stability and those with instability, mainly concerning clinical and pathological features, but also in regard to immunological microenvironment, correlation with prognostic value, and responses to treatment. In particular, gastric cancer with microsatellite instability constitutes a small but relevant subgroup associated with older age, female sex, distal stomach location, and lower number of lymph-node metastases. Emerging data attribute to microsatellite instability status a favourable prognostic meaning, whereas the poor outcomes reported after perioperative chemotherapy administration suggest a detrimental role of cytotoxic drugs in this gastric cancer subgroup. The strong immunogenicity and the widespread expression of immune-checkpoint ligands make microsatellite instability subtype more vulnerable to immunotherapeutic approach, e.g., with anti-PD-L1 and anti-CTLA4 antibodies. Since gastric cancer with microsatellite instability shows specific features and clinical behaviour not overlapping with microsatellite stable disease, microsatellite instability test might be suitable for inclusion in a diagnostic setting for all tumour stages to guarantee the most targeted and effective treatment to every patient.     

The role of key residues in structure, function, and stability of cytochrome-c

Cytochrome-c (cyt-c), a multi-functional protein, plays a significant role in the electron transport chain, and thus is indispensable in the energy-production process. Besides being an important component in apoptosis, it detoxifies reactive oxygen species. Two hundred and eighty-five complete amino acid sequences of cyt-c from different species are known. Sequence analysis suggests that the number of amino acid residues in most mitochondrial cyts-c is in the range 104?±?10, and amino acid residues at only few positions are highly conserved throughout evolution. These highly conserved residues are Cys14, Cys17, His18, Gly29, Pro30, Gly41, Asn52, Trp59, Tyr67, Leu68, Pro71, Pro76, Thr78, Met80, and Phe82. These are also known as “key residues”, which contribute significantly to the structure, function, folding, and stability of cyt-c. The three-dimensional structure of cyt-c from ten eukaryotic species have been determined using X-ray diffraction studies. Structure analysis suggests that the tertiary structure of cyt-c is almost preserved along the evolutionary scale. Furthermore, residues of N/C-terminal helices Gly6, Phe10, Leu94, and Tyr97 interact with each other in a specific manner, forming an evolutionary conserved interface. To understand the role of evolutionary conserved residues on structure, stability, and function, numerous studies have been performed in which these residues were substituted with different amino acids. In these studies, structure deals with the effect of mutation on secondary and tertiary structure measured by spectroscopic techniques; stability deals with the effect of mutation on T _m (midpoint of heat denaturation), ?G _D (Gibbs free energy change on denaturation) and folding; and function deals with the effect of mutation on electron transport, apoptosis, cell growth, and protein expression. In this review, we have compiled all these studies at one place. This compilation will be useful to biochemists and biophysicists interested in understanding the importance of conservation of certain residues throughout the evolution in preserving the structure, function, and stability in proteins.     

The role of the autonomic nervous system in mediating pancreatic endocrine responses to arginine in the calf

The release of insulin which occurred in response to arginine, in the conscious calf, differed from that which occurs in response to glucose in that it was not significantly affected by either adrenergic or muscarinic blocking agents. Release of pancreatic glucagon was reduced by pretreatment with phentolamine.     

The role of the autonomic nervous system in mediating pancreatic endocrine responses to arginine in the calf

Summary The release of insulin which occurred in response to arginine, in the conscious calf, differed from that which occurs in response to glucose in that it was not significantly affected by either adrenergic or muscarinic blocking agents. Release of pancreatic glucagon was reduced by pretreatment with phentolamine.This work was supported by the British Diabetic Association. It is a particular pleasure to acknowledge the skilled assistance provided by Messrs P. M. M. Bircham and G. P. McGregor.