Selenium and selenoproteins in mammals: extraordinary,essential, enigmatic

Selenium (Se), once known only for its potential toxicity, is now well established as an essential trace element for mammals. Insufficient Se intake predisposes to and manifests in a variety of diseases. Recent studies have proven that it is the synthesis of selenocysteine (Sec)-containing proteins, designated selenoproteins, which represents an essential prerequisite for regular development and a long and healthy life. New transgenic mouse models analysing those selenoproteins with proven enzymatic functions displayed particular phenotypes and highlighted essential Se-dependent processes in development, growth or against specific challenges. While there is a growing molecular understanding of and general agreement on the importance of sufficiently high Se intake and undisturbed selenoprotein biosynthesis, many of the recently identified selenoproteins are still uncharacterised, and the effects and consequences of supra-physiological Se dosages are not biochemically understood. With the recent definition of the human and mouse selenoproteomes and a growing number of available tools, the Se field is now geared for a great leap forward. Se biology has already broadened our knowledge about the genetic code and about protein translation. It now holds great promises also for a better understanding of some key aspects of cancer, inflammation, fertility and prevention of age-associated diseases.Received 17 March 2004; received after revision 29 April 2004; accepted 27 May 2004     

Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1^606-618). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1^759-766). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin^455-473). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.Received 12 January 2004; received after revision 23 February 2004; accepted 31 March 2004     

Kank proteins: structure, functions and diseases

The Kank family of proteins, Kank1–Kank4, are characterized by their unique structure, coiled-coil motifs in the N-terminal region, and ankyrin-repeats in the C-terminal region, with an additional motif, the KN motif, at the N-terminus. Kank1 was obtained by positional cloning of a tumor suppressor gene in renal cell carcinoma, while the other members were found by homology search. The family is involved in the regulation of actin polymerization and cell motility through signaling pathways containing PI3K/Akt and/or unidentified modulators/effectors. Their relationship to diseases such as cancer, and to neuronal and developmental disorders, will be an important subject of future study.     

Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications

Apoptosis is a morphologically distinct form of cell death. It is executed and regulated by several groups of proteins. Bcl-2 family proteins are the main regulators of the apoptotic process acting either to inhibit or promote it. More than 20 members of the family have been identified so far and most have two or more isoforms. Alternative splicing is one of the major mechanisms providing proteomic complexity and functional diversification of the Bcl-2 family proteins. Pro- and anti-apoptotic Bcl-2 family members should function in harmony for the regulation of the apoptosis machinery, and their relative levels are critical for cell fate. Any mechanism breaking down this harmony by changing the relative levels of these antagonistic proteins could contribute to many diseases, including cancer and neurodegenerative disorders. Recent studies have shown that manipulation of the alternative splicing mechanisms could provide an opportunity to restore the proper balance of these regulator proteins. This review summarises current knowledge on the alternative splicing products of Bcl-2-related genes and modulation of splicing mechanisms as a potential therapeutic approach.Received 5 January 2004; received after revision 31 March 2004; accepted 6 April 2004     

Catalysis in abiotic structured media: an approach to selective synthesis of biopolymers

Micro- and nanoenvironments formed by amphiphile self-assembled structures, water-ice lattices and minerals have well-defined, repeating, chemical and physical properties that can be used for selective synthesis of biopolymers, such as RNAs and proteins. The advances made in the development of polymerization supported by these micro- and nanosystems are reviewed here. In particular, it is shown that these systems promote non-enzymatic biopolymerization, yielding long polymers whose sequence composition is determined by the interactions between monomers and the supporting environment. When used to compartmentalize enzymatic biopolymerization, micro- and nanostructures allow the implementation of molecular selection and evolution schemes, which are difficult in homogeneous medium, yielding very active molecules. Thus, micro- and nanoenvironment approaches to the synthesis and selection of biopolymers could be developed into a new biotechnological tool for the production of biopolymers with novel functions.Received 3 August 2004; received after revision 6 October 2004; accepted 21 October 2004     

Lipopolysaccharide-binding molecules: transporters, blockers and sensors

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, can be beneficial to the host by activating the innate immune system, or harmful, by inducing inflammation, disseminated intravascular coagulation, multiple organ failure, shock and often death. On the bacteria, and in host biological fluids and cells, LPS is never free but constantly attached to cognate-binding proteins. Understanding how LPS is transported and further recognized by sensors able to deliver a signal, or by inactivating molecules able to neutralize its biological effects, is an important goal. This review describes the large panel of peptides and proteins reported to associate with LPS, and provides information on their origin, their structure and the location of amino acid residues involved in their interaction with LPS. A better understanding of the mode of recognition of LPS by cognate proteins prompted many laboratories to design on a rational basis synthetic molecules which can be used to detect low amounts of endotoxin, or to act as efficient blockers of in vitro and in vivo responses to LPS.Received 15 January 2004; received after revision 20 February 2004; accepted 25 February 2004     

Regulation of class V myosin

Class V myosin (myosin-5) is a molecular motor that functions as an organelle transporter. The activation of myosin-5′s motor function has long been known to be associated with a transition from the folded conformation in the off-state to the extended conformation in the on-state, but only recently have we begun to understand the underlying mechanism. The globular tail domain (GTD) of myosin-5 has been identified as the inhibitory domain and has recently been shown to function as a dimer in regulating the motor function. The folded off-state of myosin-5 is stabilized by multiple intramolecular interactions, including head–GTD interactions, GTD–GTD interactions, and interactions between the GTD and the C-terminus of the first coiled-coil segment. Any cellular factor that affects these intramolecular interactions and thus the stability of the folded conformation of myosin-5 would be expected to regulate myosin-5 motor function. Both the adaptor proteins of myosin-5 and Ca²⁺ are potential regulators of myosin-5 motor function, because they can destabilize its folded conformation. A combination of these regulators provides a versatile scheme in regulating myosin-5 motor function in the cell.     

The evolution of domain arrangements in proteins and interaction networks

Proteins are composed of domains, which are conserved evolutionary units that often also correspond to functional units and can frequently be detected with reasonable reliability using computational methods. Most proteins consist of two or more domains, giving rise to a variety of combinations of domains. Another level of complexity arises because proteins themselves can form complexes with small molecules, nucleic acids and other proteins. The networks of both domain combinations and protein interactions can be conceptualised as graphs, and these graphs can be analysed conveniently by computational methods. In this review we summarise facts and hypotheses about the evolution of domains in multi-domain proteins and protein complexes, and the tools and data resources available to study them.Received 20 September 2004; received after revision 23 October 2004; accepted 1 November 2004     

Protein profiling of 3T3-L1 adipocyte differentiation and (tumor necrosis factor α-mediated) starvation

The increased incidence of obesity and related disorders in Western societies requires a thorough understanding of the adipogenic process. Data at the protein level of this process are scarce. Therefore we performed a proteome analysis of differentiating and starving 3T3-L1 cells using two-dimensional gel electrophoresis combined with mass spectrometry. Effects of different starvation conditions were examined by subjecting 3T3-L1 adipocytes to caloric restriction, either in the absence or the presence of the lipolysis inducer tumor necrosis factor-. Ninety-three differentially expressed proteins were found during differentiation and starvation of 3T3-L1 cells, 50 of which were identified. GenMAPP/MAPP-finder software revealed a non-reciprocal regulation of the glycolytic pathway during 3T3-L1 differentiation followed by starvation. Furthermore, proteins involved in growth regulation, cytoskeletal rearrangements and protein modification, 16 of which have not been described before in 3T3-L1 cells, were identified. In conclusion, our data provide valuable information for further understanding of the adipogenic process.Received 9 November 2004; received after revision 21 December 2004; accepted 28 December 2004     

Hsp70 chaperones: Cellular functions and molecular mechanism

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.Received 21 October 2004; received after revision 24 November 2004; accepted 6 December 2004     

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     

The muscle ultrastructure: a structural perspective of the sarcomere

Muscle ultrastructure is characterised by a complex arrangement of many protein-protein interactions. The sarcomere is the basic repeating unit of muscle, formed by two transverse filament systems: the thick and thin filaments. While actin and myosin are the main contractile elements of the sarcomere, other proteins act as scaffolds, control ultrastructure composition, regulate muscle contraction, and transmit tension between sarcomeres and hence to the whole myofibril. Elucidation of the structures of muscle proteins by X-ray crystallography and nuclear magnetic resonance spectroscopy has been essential in understanding muscle contraction, enabling us to relate biological to structural information. These structures reveal how components of the muscle interact, how different factors influence conformational changes within these proteins, and how mutant muscle proteins may interfere with the regulatory fine-tuning of the contractile machinery, hence leading to disease in some cases. Here, structures solved within the sarcomere have been reviewed in order to put the numerous components into context.Received 28 June 2004; received after revision 25 July 2004; accepted 28 July 2004     

Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement

Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.These authors contributed equally to this work.Received 26 October 2004; received after revision 27 December 2004; accepted 6 January 2005 Available online 09 March 2005     

Cellular responses to mild heat stress

Since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock proteins that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1–2°C during febrile diseases. Here we provide information that mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells.Received 14 May 2004; received after revision 2 August 2004; accepted 16 August 2004     

Formation of the B cell synapse: retention or recruitment?

Interaction of B cells with membrane antigen results in the formation of the B cell synapse: the B cell receptor (BCR) and antigen concentrate in the contact zone while CD45/B220 and the phosphatase SHP-1 are excluded. This study shows that, unlike in T cells, synapse formation does not require active transport processes (while subsequent antigen extraction and IgM downregulation do). The synapse architecture depends on the available protein ligands in the contact zone. Thus Syk, IgM and Fc receptor accumulation require the presence of ITAM-bearing BCRs, membrane antigen and membrane (IgG-containing) immune complexes, respectively. Remarkably, non-bound proteins are frequently not only homogeneously distributed but excluded from the contact zone. These results suggest that proteins mainly reach the contact zone by undirected diffusion, and in order not to be expelled by molecular crowding they require capture by and fixation to a binding protein.Received 25 August 2004; received after revision 2 November 2004; accepted 17 November 2004     

Cellular microbiology of intracellular <Emphasis Type="Italic">Salmonella enterica</Emphasis>: functions of the type III secretion system encoded by <Emphasis Type="Italic">Salmonella</Emphasis> pathogenicity island 2

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.Received 11 June 2004; received after revision 8 July 2004; accepted 12 July 2004     

The plant thioredoxin system

Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cell. In plants, the thioredoxin system is particularly complex since at least 20 thioredoxin isoforms are found in the plant model Arabidopsis thaliana. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. Different pathways allowing thioredoxin reduction also coexist in the plant involving ferredoxin-thioredoxin reductase, thioredoxin reductases and the glutathione/glutaredoxin system. This review discusses the literature of plant thioredoxins with emphasis on recent findings in the field.Received 7 July 2004; received after revision 11 August 2004; accepted 17 August 2004     

The role of the <Emphasis Type="Italic">trans</Emphasis>-Golgi network in varicella zoster virus biology

The task of assembling nascent virions presents a formidable challenge to large, enveloped DNA viruses such as varicella zoster virus (VZV). After parasitising the host cells compartmentalised biosynthetic machinery, viral constituents must be brought together in appropriate proportions for packaging and export. Recent evidence places the trans-Golgi network (TGN) in an orchestrating role with respect to the assembly, envelopment and egress of herpesviruses. This role accords with known functions of the TGN in the uninfected cell. The targeting of viral glycoproteins to the TGN appears to provide a crucial platform for viral assembly. Tegument proteins, interacting with the cytoplasmic domains of glycoproteins, in turn recruit nucleocapsids to the developing supramolecular array. Molecular studies are continually refining understanding of these processes, building upon elegant electron microscopic data. Knowledge of VZVs use of endogenous trafficking pathways from the TGN sheds light on important aspects of viral behaviour in vitro and in vivo.Received 22 June 2004; received after revision 22 August 2004; accepted 25 August 2004     

Directed evolution of enzymes for biocatalysis and the life sciences

Engineering the specificity and properties of enzymes and proteins within rapid time frames has become feasible with the advent of directed evolution. In the absence of detailed structural and mechanistic information, new functions can be engineered by introducing and recombining mutations, followed by subsequent testing of each variant for the desired new function. A range of methods are available for mutagenesis, and these can be used to introduce mutations at single sites, targeted regions within a gene or randomly throughout the entire gene. In addition, a number of different methods are available to allow recombination of point mutations or blocks of sequence space with little or no homology. Currently, enzyme engineers are still learning which combinations of selection methods and techniques for mutagenesis and DNA recombination are most efficient. Moreover, deciding where to introduce mutations or where to allow recombination is actively being investigated by combining experimental and computational methods. These techniques are already being successfully used for the creation of novel proteins for biocatalysis and the life sciences.Received 8 June 2004; received after revision 22 July 2004; accepted 2 August 2004     

Tumor suppressor C-RASSF proteins

Human genome has ten genes that are collectedly called Ras association domain family (RASSF). RASSF is composed of two subclasses, C-RASSF and N-RASSF. Both N-RASSF and C-RASSF encode Ras association domain-containing proteins and are frequently suppressed by DNA hypermethylation in human cancers. However, C-RASSF and N-RASSF are quite different. Six C-RASSF proteins (RASSF1–6) are characterized by a C-terminal coiled-coil motif named Salvador/RASSF/Hippo domain, while four N-RASSF proteins (RASSF7–10) lack it. C-RASSF proteins interact with mammalian Ste20-like kinases—the core kinases of the tumor suppressor Hippo pathway—and cross-talk with this pathway. Some of them share the same interacting molecules such as MDM2 and exert the tumor suppressor role in similar manners. Nevertheless, each C-RASSF protein has distinct characters. In this review, we summarize our current knowledge of how C-RASSF proteins play tumor suppressor roles and discuss the similarities and differences among C-RASSF proteins.