Recent structural studies of carbohydrate-binding modules

Carbohydrate-binding modules (CBMs) are found in many carbohydrate-active enzymes. CBMs bind to a range of polysaccharides, their primary function being to increase the catalytic efficiency of the carbohydrate-active enzymes against soluble and/or insoluble substrates. CBMs bind to their target ligands with high specificities and affinities. Thus, CBM systems are excellent models to study the mechanism of protein-carbohydrate interaction. To date, CBMs have been classified into 45 different families and many structural and functional studies have been reported. At present, three-dimensional structures of CBMs from 31 different families have been determined. These structures demonstrate that the fold most commonly found in CBMs is the β-sandwich. In the past few years, about 10 new structures from different families have been reported. These enable detailed classification of CBM structures. This article reviews recent structural and functional studies of CBMs and discusses the sub-classification of β-sandwich CBMs. Received 28 April 2006; received after revision 12 July 2006; accepted 14 September 2006     

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate. Received 7 November 2005; received after revision 7 January 2006; accepted 13 January 2006     

The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones

DnaJ/Hsp40 (heat shock protein 40) proteins have been preserved throughout evolution and are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of chaperone proteins, Hsp70s. Because the ATP hydrolysis is essential for the activity of Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. DnaJ/Hsp40 proteins all contain the J domain through which they bind to Hsp70s and can be categorized into three groups, depending on the presence of other domains. Six DnaJ homologs have been identified in Escherichia coli and 22 in Saccharomyces cerevisiae. Genome-wide analysis has revealed 41 DnaJ/Hsp40 family members (or putative members) in humans. While 34 contain the typical J domains, 7 bear partially conserved J-like domains, but are still suggested to function as DnaJ/ Hsp40 proteins. DnaJA2b, DnaJB1b, DnaJC2, DnaJC20, and DnaJC21 are named for the first time in this review; all other human DnaJ proteins were dubbed according to their gene names, e.g. DnaJA1 is the human protein named after its gene DNAJA1. This review highlights the progress in studying the domains in DnaJ/Hsp40 proteins, introduces the mechanisms by which they interact with Hsp70s, and stresses their functional diversity. Received 27 April 2006; received after revision 5 June 2006; accepted 19 July 2006     

Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins

A subclass of aquaporin (AQP) water channels, termed aquaglyceroporins, are also able to transport glycerol and perhaps urea and other small solutes. Although extensive data exist on the physiological roles of aquaporin-facilitated water transport, until recently the biological significance of glycerol transport by the mammalian aquaglyceroporins has been unknown. There is now compelling evidence for involvement of aquaglyceroporin- facilitated glycerol transport in skin hydration and fat cell metabolism. Mice deficient in AQP3 have dry skin, reduced skin elasticity and impaired epidermal biosynthesis. Mice lacking AQP7 manifest progressive adipocyte fat accumulation and hypertrophy. These skin and fat phenotypes are attributable to impaired glycerol transport. A potential implication of these findings is the possibility of modulation of aquaglyceroporin expression or function in the therapy of skin diseases and obesity. Received 20 January 2006; received after revision 21 February 2006; accepted 20 March 2006     

Class III adenylyl cyclases: molecular mechanisms of catalysis and regulation

Class III adenylyl cyclases are the most abundant type of cyclic AMP-producing enzymes. The adjustment of the cellular levels of this second messenger is achieved by a variety of regulatory mechanisms which couple signals to adenylyl cyclase activity. Because of the divergent nature of stimuli which impinge on these enzymes, highly individualized class III adenylyl cyclases have evolved in metazoans, eukaryotic unicells and bacteria. Regulation usually exploits the dimeric structure of the catalyst, whose active centres form at the dimer interface. The fold of the catalytic domains and the basic catalytic mechanisms are similar in all class III adenylyl cyclases, and substrate binding generally closes the active site by an induced-fit mechanism. Regulatory inputs can result in dramatic rearrangements of the catalytic domains within the dimer, which often are based on rotational movements. Received 13 February 2006; received after revision 16 March 2006; accepted 20 April 2006     

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.Received 15 June 2004; received after revision 26 July 2004; accepted 2 August 2004     

Cardiolipin, the heart of mitochondrial metabolism

Cardiolipin is a unique phospholipid, which is almost exclusively localized in the mitochondrial inner membrane where it is synthesized from phosphatidylglycerol and cytidinediphosphate-diacylglycerol. After primary synthesis, the mature acyl chain composition of cardiolipin is achieved by at least two remodeling mechanisms. In the mitochondrial membrane cardiolipin plays an important role in energy metabolism, mainly by providing stability for the individual enzymes and enzyme complexes involved in energy production. Moreover, cardiolipin is involved in different stages of the mitochondrial apoptotic process and in mitochondrial membrane dynamics. Cardiolipin alterations have been described in various pathological conditions. Patients suffering from Barth syndrome have an altered cardiolipin homeostasis caused by a primary deficiency in cardiolipin remodeling. Alterations in cardiolipin content or composition have also been reported in more frequent diseases such as diabetes and heart failure. In this review we provide an overview of cardiolipin metabolism, function and its role in different pathological states. Received 16 January 2008; received after revision 26 February 2008; accepted 26 March 2008     

The life cycle of neutral lipids: synthesis, storage and degradation

Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) form the group of neutral lipids. Whereas TAGs are present in all types of cell, the occurrence of SEs in prokaryotes is questionable, and the presence of WEs as storage molecules is restricted to plants and a few bacteria. Here, we summarize recent knowledge on the formation, storage and degradation of TAGs and SEs in various cell types. We describe the biochemical pathways involved in TAG and SE synthesis and discuss the subcellular compartmentation of these processes. Recently, several novel enzymes governing the metabolism of storage lipids have been identified and characterized. Regulatory aspects of neutral lipid storage are just beginning to be understood. Finally, we describe consequences of defects in neutral lipid metabolism. Since severe diseases like atherosclerosis, obesity and type 2 diabetes are caused by lipid accumulation, mechanisms underlying neutral lipid synthesis, depot formation and mobilization are of major interest for curing such diseases that are increasingly associated with modern civilization. Received 18 January 2006; received after revision 7 March 2006; accepted 16 March 2006     

Magnetic resonance and the human brain: anatomy, function and metabolism

The introduction and development, over the last three decades, of magnetic resonance (MR) imaging and MR spectroscopy technology for in vivo studies of the human brain represents a truly remarkable achievement, with enormous scientific and clinical ramifications. These effectively non-invasive techniques allow for studies of the anatomy, the function and the metabolism of the living human brain. They have allowed for new understandings of how the healthy brain works and have provided insights into the mechanisms underlying multiple disease processes which affect the brain. Different MR techniques have been developed for studying anatomy, function and metabolism. The primary focus of this review is to describe these different methodologies and to briefly review how they are being employed to more fully appreciate the intricacies associated with the organ, which most distinctly differentiates the human species from the other animal forms on earth. Received 1 November 2005; received after revision 11 January 2006; accepted 25 January 2006     

Selectivity determinants of the aldose and aldehyde reductase inhibitor-binding sites

Aldose reductase and aldehyde reductase belong to the aldo-keto reductase superfamily of enzymes whose members are responsible for a wide variety of biological functions. Aldose reductase has been identified as the first enzyme involved in the polyol pathway of glucose metabolism which converts glucose into sorbitol. Glucose over-utilization through the polyol pathway has been linked to tissue-based pathologies associated with diabetes complications, which make the development of a potent aldose reductase inhibitor an obvious and attractive strategy to prevent or delay the onset and progression of the complications. Structural studies of aldose reductase and the homologous aldehyde reductase in complex with inhibitor were carried out to explain the difference in the potency of enzyme inhibition. The aim of this review is to provide a comprehensive summary of previous studies to aid the development of aldose reductase inhibitors that may have less toxicity problems than the currently available ones. Received 4 December 2006; received after revision 12 February 2007; accepted 20 April 2007     

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M _r of 25 – 30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology. Received 21 November 1997; received after revision 10 March 1998; accepted 10 March 1998     

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments. Received 21 November 2005; received after revision 20 January 2006; accepted 2 February 2006 An erratum to this article is available at .     

Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer

The fraction of pyruvate dehydrogenase complex (PDC) in the active form is reduced by the activities of dedicated PD kinase isozymes (PDK1, PDK2, PDK3 and PDK4). Via binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2 60mer), PDK rapidly access their E2-bound PD substrate. The E2-enhanced activity of the widely distributed PDK2 is limited by dissociation of ADP from its C-terminal catalytic domain, and this is further slowed by pyruvate binding to the N-terminal regulatory (R) domain. Via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation. Activation of PDC by synthetic PDK inhibitors binding at the pyruvate or lipoyl binding sites decreased damage during heart ischemia and lowered blood glucose in insulin-resistant animals. PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate. Received 25 August 2006; received after revision 20 November 2006; accepted 20 December 2006     

Regulation of cell adhesion by PP2A and SV40 small tumor antigen: An important link to cell transformation

The serine/threonine protein phosphatase 2A (PP2A) represents a large family of highly conserved heterotrimeric enzymes. Their critical importance in cell homeostasis is underlined by the fact that they are targets of natural toxins like the tumor promoter okadaic acid, and of simian virus 40 small tumor antigen (SV40 small t), a viral protein known to promote cell transformation. Furthermore, mutated or lower expression levels of PP2A subunits have been found in certain cancers. One major known event in PP2A-dependent cell transformation is the alteration of key signaling pathways that control cell growth and survival. In this review, we focus on how PP2A enzymes also affect cell adhesion and cytoskeletal dynamics, the disruption of which is linked to loss of cell polarity, increased cell motility and invasiveness. We also examine how those various pathways participate in the transforming activity of SV40 small t. Received 29 June 2006; received after revision 3 August 2006; accepted 20 September 2006     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

Phytanic acid: production from phytol, its breakdown and role in human disease

Phytanic acid is a branched-chain fatty acid that accumulates in a variety of metabolic disorders. High levels of phytanic acid found in patients can exceed the millimolar range and lead to severe symptoms. Degradation of phytanic acid takes place by α-oxidation inside the peroxisome. A deficiency of its breakdown, leading to elevated levels, can result from either a general peroxisomal dysfunction or from a defect in one of the enzymes involved in α-oxidation. Research on Refsum disease, belonging to the latter group of disorders and characterized by a deficiency of the first enzyme of α-oxidation, has extended our knowledge of phytanic acid metabolism and pathology of the disease greatly over the past few decades. This review will centre on this research on phytanic acid: its origin, the mechanism by which its α-oxidation takes place, its role in human disease and the way it is produced from phytol. Received 4 October 2005; received after revision 24 February 2006; accepted 26 April 2006     

Turning points in the evolution of peroxidase–catalase superfamily: molecular phylogeny of hybrid heme peroxidases

Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase–catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase–catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.     

Regulation of insulin receptor function

Resistance to the biological actions of insulin contributes to the development of type 2 diabetes and risk of cardiovascular disease. A reduced biological response to insulin by tissues results from an impairment in the cascade of phosphorylation events within cells that regulate the activity of enzymes comprising the insulin signaling pathway. In most models of insulin resistance, there is evidence that this decrement in insulin signaling begins with either the activation or substrate kinase activity of the insulin receptor (IR), which is the only component of the pathway that is unique to insulin action. Activation of the IR can be impaired by post-translational modifications of the protein involving serine phosphorylation, or by binding to inhibiting proteins such as PC-1 or members of the SOCS or Grb protein families. The impact of these processes on the conformational changes and phosphorylation events required for full signaling activity, as well as the role of these mechanisms in human disease, is reviewed in this article. Received 3 August 2006; received after revision 1 December 2006; accepted 8 January 2007     

Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism?

Betaine homocysteine methyltransferase (BHMT), a Zn²⁺-dependent thiolmethyltransferase, contributes to the regulation of homocysteine levels, increases in which are considered a risk factor for cardiovascular diseases. Most plasma homocysteine is generated through the liver methionine cycle, in which BHMT metabolizes approximately 25% of this non-protein amino acid. This process allows recovery of one of the three methylation equivalents used in phosphatidylcholine synthesis through transmethylation, a major homocysteine-producing pathway. Although BHMT has been known for over 40 years, the difficulties encountered in its isolation precluded detailed studies until very recently. Thus, the last 10 years, since the sequence became available, have yielded extensive structural and functional data. Moreover, recent findings offer clues for potential new functions for BHMT. The purpose of this review is to provide an integrated view of the knowledge available on BHMT, and to analyze its putative roles in other processes through interactions uncover to date. Received 26 May 2006; received after revision 3 July 2006; accepted 24 August 2006     

Family I.3 lipase: bacterial lipases secreted by the type I secretion system

Based on the classification of bacterial lipolytic enzymes, family I.3 lipase is a member of the large group of Gram-negative bacterial true lipases. This lipase family is distinguished from other families not only by the amino acid sequence, but also by the secretion mechanism. Lipases of family I.3 are secreted via the well-known type I secretion system. Like most of proteins secreted via this system, family I.3 lipases are composed of two domains with distinct yet related functions. Recent years have seen an increasing amount of research on this lipase family, in terms of isolation, secretion mechanism, as well as biochemical and biophysical studies. This review describes our current knowledge on the structure-function relationships of family I.3 lipase, with an emphasis on its secretion mechanism. Received 18 April 2006; received after revision 3 July 2006; accepted 24 August 2006