FHA-RING ubiquitin ligases in cell division cycle control

Despite the common occurrence of forkhead associated (FHA) phosphopeptide-binding domains and really interesting new gene (RING) E3 ubiquitin ligase domains, gene products containing both an N-terminal FHA domain and C-terminal RING domain constitute a highly distinctive intersection. Characterized FHA-RING ligases include the two vertebrate proteins, Checkpoint with FHA and RING (Chfr) and RING finger 8 (Rnf8), as well as three fungal proteins, Defective in mitosis (Dma1), Chf1 and Chf2. These FHA-RING ligases play roles in negative regulation of the cell division cycle, apparently by coupling protein phosphorylation events to specific ubiquitylation of target proteins. Here, the available data on upstream and downstream regulation of and by FHA-RING ligases are reviewed. Received 24 April 2008; received after revision 18 June 2008; accepted 20 June 2008     

Integrase, LEDGF/p75 and HIV replication

HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy. Received 25 November 2007; received after revision 7 January 2008; accepted 10 January 2008     

The perspectives of studying multi-domain protein folding

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding. Received 3 December 2008; accepted 23 December 2008     

hShroom1 links a membrane bound protein to the actin cytoskeleton

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with β-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton. D. E. Dye, S. Karlen: These authors contributed equally to this work. Received 09 October 2008; received after revision 23 November 2008; accepted 09 December 2008     

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 September 2008; received after revision 17 December 2008; accepted 06 January 2009     

CCCTC-binding factor: to loop or to bridge

Eukaryotic genomes have complex spatial organization in the nucleus. The factors and the mechanisms involved in this organization remain an enigma. Among the many proteins implicated in such a role, the ubiquitous Zn-finger protein CTCF stands out. Here we summarize the evidence placing CTCF in the enviable position of a master organizer of the genome. CTCF can form loops in cis, and can bridge sequences located on different chromosomes in trans. The thousands of CTCF binding sites, identified in recent genome-wide localization studies, and their distribution along the genome further support a crucial role of CTCF as a chromatin organizer. Received 10 October 2008; received after revision 11 December 2008; accepted 16 December 2008     

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     

Recombinant expression of perchloric acid-soluble protein reduces cell proliferation

Perchloric acid-soluble protein (PSP) may play an important role in the regulation of cellular physiological functions because it has been highly conserved throughout evolution; however, this role has not been well elucidated. In previous reports, we suggested that PSP regulates cell proliferation. In this study, we examined the effect of PSP expression on proliferation of the normal rat kidney cell line NRK-52E, the rat hepatocyte cell line RLN-10, and the rat hepatoma cell line dRLh-84. Cells transfected with pcDNA-sense-PSP (pcDNA-S-PSP) over-expressed PSP mRNA and protein, and cell proliferation of the transfected cells was suppressed compared with that of cells transfected with pcDNA-empty (pcDNA-E). Cell viability of pcDNA-S-PSP-transfected cells was similar to that of pcDNA-E-transfected cells. Thus, over-expression of PSP suppresses cell proliferation without any influence on cell viability. These findings are the first to report an inhibitory activity of PSP on cell proliferation. Received 27 April 2001; received after revision 8 June 2001; accepted 8 June 2001     

Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27

G₁ phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.     

From immune response to cancer: a spot on the low molecular weight protein tyrosine phosphatase

Reversible tyrosine phosphorylation is a key posttranslational regulatory modification of proteins in all eukaryotic cells in normal and pathological processes. Recently a pivotal janus-faced biological role of the low molecular weight protein tyrosine phosphatase (LMWPTP) has become clear. On the one hand this enzyme is important in facilitating appropriate immune responses towards infectious agents, on the other hand it mediates exaggerated inflammatory responses toward innocuous stimuli. The evidence that LMWPTP plays a role in oncological processes has added a promising novel angle. In this review we shall focus on the regulation of LMWPTP enzymatic activity of signaling pathways of different immunological cells, the relation between genetic polymorphism of LMWPTP and predisposition to some type of inflammatory disorders and the contribution of this enzyme to cancer cell onset, growth and migration. Therefore, the LMWPTP is an interesting target for pharmacological intervention, thus modifying both inappropriate cellular immune responses and cancer cell aggressiveness. Received 15 August 2008; received after revision 06 October 2008; accepted 14 October 2008     

Coilin levels and modifications influence artificial reporter splicing

Cajal bodies (CBs) and Gems are nuclear domains that contain factors responsible for spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis. The marker protein for CBs is coilin. In addition to snRNPs, coilin and other factors, canonical CBs contain the survivor of motor neuron protein (SMN). SMN can also localize to Gems. Considering the important role that coilin plays in the formation and composition of CBs, we tested the splicing efficiency of several cell lines that vary in regards to coilin level and modification using an artificial reporter substrate. We found that cells with both hypomethylated coilin and Gems are more efficient at reporter splicing compared to cells in which SMN localizes to CBs. In contrast, coilin reduction, which induces Gem formation, decreases cell proliferation and artificial reporter splicing. These findings demonstrate that coilin modifications or levels impact artificial reporter splicing, possibly by influencing snRNP biogenesis. Received 26 December 2007; received after revision 5 February 2008; accepted 7 February 2008     

S100A6 protein: functional roles

S100A6 protein belongs to the A group of the S100 protein family of Ca²⁺-binding proteins. It is expressed in a limited number of cell types in adult normal tissues and in several tumor cell types. As an intracellular protein, S100A6 has been implicated in the regulation of several cellular functions, such as proliferation, apoptosis, the cytoskeleton dynamics, and the cellular response to different stress factors. S100A6 can be secreted/released by certain cell types which points to extracellular effects of the protein. RAGE (receptor for advanced glycation endproducts) and integrin β1 transduce some extracellular S100A6’s effects. Dosage of serum S100A6 might aid in diagnosis in oncology.