Molecular cloning and primary structure of human glial fibrillary acidic protein.

Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, we isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities--particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain--with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.     

Identifying protein interactors in gonadotropin action

Systems biology integrates a variety of diverse approaches to the study of the cellular pathways comprised of protein networks. Following an initial ligand-receptor binding event, transduction of the signal is modified in a variety of ways via downstream protein interactions. Protein interactions can occur between two proteins or, alternatively, an interaction between two proteins can be facilitated by an adapter protein. Protein interactions can affect the spatial and temporal distribution of ligand-receptor complexes in cells, attenuating or prolonging signaling. With regard to gonadotropin receptors, protein interactions have been primarily studied in terms of desensitization and termination of signal transduction, or for their role in trafficking. The purpose of this review is to describe protein interactions that mediate gonadotropin receptor functions and to highlight some emerging interactions, as well as some of the caveats inherent in the attempt to uncover these pathways.     

Human cytomegalovirus latency alters the cellular secretome, inducing cluster of differentiation (CD)4+ T-cell migration and suppression of effector function

After primary infection, human cytomegalovirus (HCMV) persists as a life-long latent infection, with host immunosuppression often resulting in clinical reactivation. During lytic infection, major changes in the expression of secreted cellular proteins (the secretome) occur that have profound effects on host-cell interactions, particularly at the level of the host immune response. In contrast, little is known about changes in the secretome that accompany latent infection, yet this is likely to be of major importance for the life-long carriage of this persistent human pathogen in the face of constant immunosurveillance. We have analyzed the secretome of cells carrying latent HCMV and have identified changes in several secreted cellular proteins known to be involved in regulation of the immune response and chemoattraction. Here, we show that a latency-associated increase in CC chemokine ligand (CCL)8 results in the recruitment of cluster of differentiation (CD)4(+) T cells to supernatants from latently infected CD34(+) cells but that these latent supernatants, also rich in immunosuppressive factors, inhibit cytokine secretion and cytotoxicity of HCMV-specific T-helper (Th)1 CD4(+) T cells. These results identify a strategy by which sites of latent HCMV can firstly recruit CD4(+) T cells and then inhibit their antiviral effector functions, thereby aiding the maintenance of latent infection in the face of the host immune response.     

Heat shock proteins as regulators of the immune response

Until recently, heat shock proteins (also known as heat stress proteins) have mostly been regarded as intracellular molecules that mediate a range of essential housekeeping and cytoprotective functions. However, interest in their role as intercellular signalling molecules has been fuelled by the observations that these molecules can be released and are present in the extracellular environment under physiological conditions. They can elicit cytokine production by, and adhesion molecule expression of, a range of cell types, and they can deliver maturation signals and peptides to antigen presenting cells through receptor-mediated interactions. These functions suggest that heat shock proteins could be immunoregulatory agents with potent and widely-applicable therapeutic uses. Furthermore, the induction of self heat shock protein immune reactivity can attenuate autoimmunity and delay transplant rejection, and heat shock proteins derived from tumours and pathogens can elicit specific, protective immunity. This review will focus on this rapidly evolving area of heat shock protein biology.     

Manipulation of Host Microtubule Networks by Viral Microtubule-Associated Proteins

Diverse DNA and RNA viruses utilize cytoskeletal networks to efficiently enter, replicate, and exit the host cell, while evading host immune responses. It is well established that the microtubule (MT) network is commonly hijacked by viruses to traffic to sites of replication after entry and to promote egress from the cell. However, mounting evidence suggests that the MT network is also a key regulator of host immune responses to infection. At the same time, viruses have acquired mechanisms to manipulate and/or usurp MT networks to evade these immune responses. Central to most interactions of viruses with the MT network are virally encoded microtubule-associated proteins (MAPs) that bind to MTs directly or indirectly. These MAPs associate with MTs and other viral or cellular MAPs to regulate various aspects of the MT network, including MT dynamics, MT-dependent transport via motor proteins such as kinesins and dyneins, and MT-dependent regulation of innate immune responses. In this review, we examine how viral MAP interactions with the MT network facilitate viral replication and immune evasion.     

Mechanisms of disease: protease functions in intestinal mucosal pathobiology

Of all our organ systems, the gastrointestinal tract contains the highest levels of endogenous and exogenous proteases (also known as proteinases and peptidases); however, our understanding of their functions and interactions within the gastrointestinal tract is restricted largely to nutrient digestion. The gut epithelium is a sensor of the luminal environment, not only controlling digestive, absorptive and secretory functions, but also relaying information to the mucosal immune, vascular and nervous systems. These functions involve a complex array of cell types that elaborate growth factors, cytokines and extracellular matrix (ECM) proteins, the activity and availability of which are regulated by proteases. Proteolytic activity must be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological gastrointestinal conditions. Moreover, enteric microbes and parasites can hijack proteolytic pathways through 'pathogen host mimicry'. Understanding how the protease balance is maintained and regulated in the intestinal epithelial cell microenvironment and how proteases contribute to physiological and pathological outcomes will undoubtedly contribute to the identification of new potential therapeutic targets for gastrointestinal diseases.     

Identification and partial characterization of six members of the kinesin superfamily in Drosophila.

Recent evidence has suggested that the principal polypeptide component of the microtubule motor protein kinesin may be a member of an extended superfamily of related motor proteins. To gain insight into how large the kinesin superfamily might be and to begin determining the potential functions in which various superfamily members might participate, we identified and partially characterized six additional members of the Drosophila kinesin superfamily. Genes encoding these proteins were identified by using the polymerase chain reaction with degenerate primers corresponding to highly conserved regions of the kinesin heavy-chain motor domain. Partial sequencing of the six genes revealed that they encode proteins that are 40-60% identical to the motor domain of the kinesin heavy-chain sequence. The cytogenetic locations as well as the developmental and tissue-specific expression patterns have been determined. The data suggest that each of these six kinesin-like proteins may have functions in a wide variety of cell types and tissues.     

Functions of red cell surface proteins

The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.     

Characterization of a class of cationic proteins that specifically interact with intermediate filaments

We describe a class of cationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types of cytoskeletal proteins. These proteins, for which the term filaggrin is introduced, are isolated from the stratum corneum of mammalian epidermis. They are species-distinct proteins; for example, rat and mouse filaggrin have different molecular weights and amino acid compositions, but are nevertheless chemically and functionally ver similar. They interact in vitro with the IF several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. Stoichiometric analyses suggest that two molecules of filaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil alpha-helical regions of the IF.     

Role of macrophages and monocytes in hepatitis C virus infections

A number of studies conducted over many years have shown that hepatitis C virus(HCV)can infect a variety of cell types.In vivo infection of monocytes,macrophages,and dendritic cells by HCV has been frequently shown by a number of researchers.These studies have demonstrated replication of HCV by detecting the presence of both negative genomic strands and a variety of non-structural HCV proteins in infected cells.In addition,analyses of genome sequences have also shown that different cell types can harbor different HCV variants.Investigators have also done preliminary studies of which cellular genes are affected by HCV infection,but there have not yet been a sufficient number of these studies to understand the effects of infection on these cells.Analyses of in vitro HCV replication have shown that monocytes,macrophages and dendritic cells can be infected by HCV from patient sera or plasma.These studies suggest that entry and cellular locations may vary between different cell types.Some studies suggest that macrophages may preferentially allow HCV genotype 1 to replicate,but macrophages do not appear to select particular hypervariable regions.Overall,these studies agree with a model where monocytes and macrophages act as an amplification system,in which these cells are infected and show few cytopathic effects,but continuously produce HCV.This allows them to produce virus over an extended time and allows its spread to other cell types.     

Identification of two novel transmembrane gamma-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich gamma-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20-24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.     

Berberine in Human Oncogenic Herpesvirus Infections and Their Linked Cancers

Human herpesviruses are known to induce a broad spectrum of diseases, ranging from common cold sores to cancer, and infections with some types of these viruses, known as human oncogenic herpesviruses (HOHVs), can cause cancer. Challenges with viral latency, recurrent infections, and drug resistance have generated the need for finding new drugs with the ability to overcome these barriers. Berberine (BBR), a naturally occurring alkaloid, is known for its multiple biological activities, including antiviral and anticancer effects. This paper comprehensively compiles all studies that have featured anti-HOHV properties of BBR along with promising preventive effects against the associated cancers. The mechanisms and pathways induced by BBR via targeting the herpesvirus life cycle and the pathogenesis of the linked malignancies are reviewed. Approaches to enhance the therapeutic efficacy of BBR and its use in clinical practice as an anti-herpesvirus drug are also discussed.     

Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin beta tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin beta tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.     

Interface between membrane biology and clinical medicine

Many enzymes that are embedded within membranes of cells are sensitive to the chemical and physical properties of the lipid components of the membrane. Because of this, the functions of these integral membrane-bound enzymes can be regulated to some extent by changes within the lipid portions of biologic membranes. That the functions of membrane-bound proteins can be manipulated by modifications of their intimate environment is not surprising. It is well known, for example, that the properties of the surrounding aqueous phase can modulate the function of proteins that are soluble in the cytosol of cells. In contrast, whereas significant changes in the chemical composition and physical properties of the aqueous portion of the cell (e.g., ionic strength and pH) are not allowed, normally tolerable fluctuations of diet appear to influence significantly the composition and properties of the lipid portions of intracellular membranes to the extent of altering the function of some membrane-bound enzymes. In addition, it appears that changes of this type can be induced by diseases that alter dietary intake and/or intermediary metabolism. In other words, it is likely that the functions of at least some integral membrane proteins can be manipulated in patients. Such manipulations may prove to be efficacious. Alternatively, the importance of diet and processes of intermediary metabolism for altering the course of certain diseases may not be fully appreciated. It is worthwhile to review, therefore, current ideas of how the lipid portion of a membrane interacts with integral proteins. The property of the lipids that appears to be most important in this regard is their viscosity. The types of manipulations of function of membrane-bound enzymes that can be achieved are illustrated by in vitro effects secondary to varying the lipids used to reconstitute pure, delipidated forms of these enzymes. The functions of pure delipidated enzymes are discussed for the hepatic drug-metabolizing enzyme uridine diphosphoglucuronyltransferase. In addition, data are presented to indicate that the function of this enzyme can be modified extensively in intact animals by changing the diet.     

Hepatocellular carcinoma: role of hepatitis B and hepatitis C viruses proteins in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer, being a common cancer type worldwide. Many aetiological factors have been related with HCC development, such as cirrhosis, hepatitis viruses and alcohol. Chronic infection with hepatitis B (HBV) and C viruses (HCV) often results in cirrhosis and enhances the probability of developing HCC. The underlying mechanisms that lead to malignant transformation of infected cells, however, remain unclear. HBV is a DNA virus that integrates into the host genome, and this integration is believed, in part, to be carcinogenic. Besides, the virus encodes a 17 kDa protein, HBx, which is known to be a causative agent in the formation of HCC. On the contrary, HCV is a RNA virus that does not integrate into the host genome but likely induces HCC through host protein interactions or via the inflammatory response to the virus. Products encoded in the HCV genome interfere with and disturb intracellular signal transduction. Some HCV proteins, such as the core protein, NS3 and NS5A, have seen to have a regulatory effect on cellular promoters, to interact with a number of cellular proteins, and to be involved in programmed-cell death modulation under certain conditions. The identification of these proteins functions in HCC development and the subsequent development of strategies to inhibit protein-protein interactions may be the first step towards reducing the chronicity and/or of the carcinogenicity of these two viruses.     

Roles of Virion-Incorporated CD162 (PSGL-1), CD43, and CD44 in HIV-1 Infection of T Cells

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4⁺ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4⁺ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4⁺ T cells.