Class Of Cellular Proteins Research Articles

Ion Channels and Ions as Therapeutic Targets and Strategies for Herpes Simplex Virus Infection

Herpes simplex virus (HSV) is a highly contagious virus that cannot be completely cured currently. Existing treatment methods are mainly nucleoside antiviral drugs, and the emergence of drug-resistant strains severely limits their use. There is an urgent need to discover antiviral drugs that act on new targets. Ion channels, a class of cellular proteins with a wide range of functions, have become critical host factors for a wide variety of viral infections. Ion channel blockers have been shown to have antiviral activity. In this study, we discuss the role of ion channels and ions in the HSV life cycle, and the potential of targeting ion channels as a novel, pharmacologically safe and wide-range antiviral treatment option.

The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.

Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors.

Crystal Structure of the SF3 Helicase from Adeno-Associated Virus Type 2

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA + class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected “arginine finger” that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA + domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA + proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.

Effect of acute and chronic lead exposure on the level of sulfhydryl groups in rat brain.

Lead is known to be a potent neurotoxic agent. The interactions of lead with proteins are considered to be at least one of the mechanisms due to which lead exerts its toxicity. In the present work we demonstrate that acute and chronic models of exposure to lead affected the level of total and protein-bound SH groups in homogenates and synaptosomes obtained from rat brains. The concentrations of SH groups were lowered significantly (P < 0.05) in both types of poisoning. Different classes of cellular proteins were considered to play a role in high affinity binding of lead to these ligands.

Defective lens fiber differentiation and pancreatic tumorigenesis caused by ectopic expression of the cellular retinoic acid-binding protein I

All-trans retinoic acid, a metabolite of retinol, is a possible morphogen in vertebrate development. Two classes of cellular proteins, which specifically bind all-trans retinoic acid, are thought to mediate its action: the nuclear retinoic acid receptors (RAR alpha, beta, gamma), and the cytoplasmic binding proteins known as cellular retinoic acid-binding proteins I and II (CRABP I and II). The function of the retinoic acid receptors is to regulate gene transcription by binding to DNA in conjunction with the nuclear retinoid X receptors (RXR alpha, beta, gamma), which in turn have 9-cis retinoic acid as a ligand. Several lines of evidence suggest that the role of the cellular retinoic acid-binding proteins is to control the concentration of free retinoic acid reaching the nucleus in a given cell. Here, we have addressed the role of the cellular retinoic acid-binding protein I in development by ectopically expressing it in the mouse lens, under the control of the alpha A-crystallin promoter. We show that this ectopic expression interferes with the development of the lens and with the differentiation of the secondary lens fiber cells, causing cataract formation. These results suggest that correct regulation of intracellular retinoic acid concentration is required for normal eye development. In addition, the generated transgenic mice also present expression of the transgene in the pancreas and develop pancreatic carcinomas, suggesting that overexpression of the cellular retinoic acid-binding protein is the cause of the tumors. These results taken together provide evidence for a role of the cellular retinoic acid-binding protein in development and cell differentiation. The relevance of these findings to the possible role of the cellular retinoic acid-binding proteins in the transduction of the retinoic acid signal is discussed.

Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

Occurrence of tyrosine sulfate in proteins – a balance sheet

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.

Constitutive synthesis of globins within most of the members of an uninduced, proliferating population of Friend erythroleukemic cells

The proteins synthesized by Friend erythroleukemic cells (clone 745), before and after they have been induced by treatment with dimethylsulfoxide to undergo overt erythroid maturation, have been analyzed by a number of biochemical methods. None of these techniques has, however, revealed any consistent observable differences (other than quantitative variations in the concentrations of particular proteins) between the “uninduced” and the “induced” cells. Thus, various classes of cellular proteins (whole cell, cytoplasmic, and nuclear) appear to be very similar in the two types of cells when analyzed by two-dimensional gel electrophoresis. Of particular interest is the finding that the strain-specific hemoglobins of DBA 2 mice (the line from which the Friend cells were derived by viral transformation) are being synthesized in both the uninduced and the induced cells but at apparently markedly different rates. A monospecific antibody preparation against purified DBA 2 mouse α- and β-globins was used to quantify the amount of these proteins in Friend cells during the course of dimethylsulfoxide-induced differentiation. Rocket gel immunoelectrophoresis, radial immunodiffusion analysis, and competitive radioimmunoassay revealed that the uninduced Friend cells contained, on the average, about 0.45 pg of hemoglobin per cell and this amount increased about 32-fold (to about 14.4 pg per cell) after 5 days of induction. Furthermore, use of the antibody preparation in indirect immunofluorescence studies revealed the constitutive synthesis of low levels of globins in virtually all of the uninduced, actively dividing, leukemia cells. These studies indicate that the standardly used benzidine staining method for detecting hemoglobins is insensitive for the detection of low levels of these proteins. The results of this study indicate that the cells in this particular leukemic population are, in reality, already “differentiated.” This suggests that the cells may represent an intermediate stage of erythroid maturation whose further progress along the normal in vivo terminal differentiation pathway has been blocked by viral transformation. Perhaps various “inducers,” such as dimethylsulfoxide, may enable these cells to partially overcome this block.

Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli minicells.

E. coli minicells lack DNA, yet they make protein, the synthesis of which is sensitive to chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA with a short half-life whose synthesis is rifamycin-sensitive and (ii) cellular mRNA with a long half-life whose synthesis is rifamycin-insensitive. These findings indicate that minicells contain normal degradative mechanisms for mRNA and point out the existence of a unique class of very stable cellular mRNA. Greater than 80% of the rifamycin-insensitive protein synthesized goes into the outer minicell membrane. Relatively stable mRNA, half-life 5.5-11.5 min, for outer membrane protein in whole cells has been reported [Hirashima et al. (1973) J. Mol. Biol. 79, 373-389]. The stability of minicell mRNA is significantly greater. This and other observations suggest that there are two functional species of mRNA for outer membrane protein perhaps in different sites in the cell. Furthermore, these studies suggest that a class of cellular proteins is synthesized in bacteria without concomitant transcription and in the absence of association with chromosomal DNA.