RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.     

Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.     

Discovering differential activation machinery of the Toll-like receptor 4 signaling pathways in MyD88 knockouts

To understand differential time activation of nuclear factor kappaB (NF-kappaB) and the temporal features of the downstream pro-inflammatory cytokines' [tumour-necrosis-factor-alpha (TNF-alpha) and IP-10] mRNA levels in myeloid differentiation primary-response protein 88 (MyD88) knockouts (KOs), I developed a computational model of the TLR4 pathway. The result suggests that the late phase expression of NF-kappaB activity observed in MyD88 KOs is possibly due to a number of novel intermediates acting along the MyD88-independent pathway. I also simulate that the TNF-alpha levels will increase at a longer time in MyD88 KOs, not previously mentioned.     

Sex-specific association between X-linked Toll-like receptor 7 with the outcomes of hepatitis C virus infection

Toll-like receptor 7 (TLR7) senses hepatitis C virus (HCV) infection and drives the host specific innate and adaptive immune response. The aim of this study was to estimate the distributions of TLR7 single nucleotide polymorphisms (SNPs), including rs179019 and rs3853839, as well as the effect of TLR7 gene variants on TLR7 mRNA expression and cytokine production in response to TLR7 agonist in vitro. TLR7 SNP genotyping was performed among a Chinese sample population of 418 patients with persistent HCV infection, 317 patients with HCV spontaneous clearance, and 989 healthy controls. TLR7 mRNA expression and TLR7-specific IFN-α and IL-6 secretion in peripheral blood mononuclear cells, derived from 60 healthy individuals in vitro, were then quantified. We identified the association of TLR7 rs3853839C allele, haplotype CC and haplotype AC (rs179019/rs3853839) with protection against HCV persistence in Chinese females (OR = 0.49, 95% CI = 0.29–0.81, P = 0.01 for rs3853839 GC; OR = 0.29, 95% CI = 0.11–0.75, P = 0.01 for rs3853839 CC; OR = 0.51, 95% CI = 0.38–0.77, P < 0.01 for haplotype CC; OR = 0.29, 95% CI = 0.10–0.88, P = 0.03 for haplotype AC). In addition, the rs3853839 CC genotype among female carriers had significantly low TLR7 mRNA expression (P = 0.006 for GG vs. CC, P = 0.021 for GC vs. CC), along with decreased IFN-α (P = 0.002 for GG vs. CC, P = 0.021 for GC vs. CC) and increased antiviral IL-6 production (P = 0.002 for GG vs. CC, P = 0.030 for GC vs. CC), after treatment with Imiquimod in vitro. The cytokine profile among rs3853839 CC genotype female carriers may indicate a pronounced protective effect against persistent HCV infection. The functional polymorphism of TLR7 rs3853839C allele was found to be sex-specific and associated with protection against HCV persistence among Chinese females, which may be due to specific IFN-α and IL-6 secretion profiles.     

StarD4-mediated translocation of 7-hydroperoxycholesterol to isolated mitochondria: Deleterious effects and implications for steroidogenesis under oxidative stress conditions

StAR family proteins, including StarD4, play a key role in steroidogenesis by transporting cholesterol (Ch) into mitochondria for conversion to pregnenolone. Using a model system consisting of peroxidized cholesterol (7α-OOH)-containing liposomes as donors, we showed that human recombinant StarD4 accelerates 7α-OOH transfer to isolated liver mitochondria, and to a greater extent than Ch transfer. StarD4 had no effect on transfer of non-oxidized or peroxidized phosphatidylcholine, consistent with sterol ring specificity. StarD4-accelerated 7α-OOH transfer to mitochondria resulted in greater susceptibility to free radical lipid peroxidation and loss of membrane potential than in a non-StarD4 control. The novel implication of these findings is that in oxidative stress states, inappropriate StAR-mediated trafficking of peroxidized Ch in steroidogenic tissues could result in damage and dysfunction selectively targeted to mitochondria.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

An anti-interferon activity shared by paramyxovirus C proteins: Inhibition of Toll-like receptor 7/9-dependent alpha interferon induction

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.     

Cationic lipids activate cellular cascades. Which receptors are involved?

Cationic lipids have been extensively used as carriers of biologically active molecules (nucleic acids, peptides and proteins) into cells. Recent data provided evidence that cationic lipids are not just inert transporters but do activate specific cellular cascades. This review illustrates these activating properties with a few examples. Cell activation raises the question of which receptors are involved. Some cationic lipids seem to satisfy specific structural requirements of Toll-like receptors (TLR4) as they activate TLR4-dependent pathways. However, cationic lipids display a large structural diversity and it is likely that they are also recognized by receptors with a broader specificity. Alternatives are proposed and discussed to explain this broad specificity.     

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies.In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.     

Cyclin-dependent kinase activating kinase/Cdk7 co-localizes with PKC-iota in human glioma cells

Cyclin-dependent kinase activating kinase (CAK) is a trimeric complex composed of cdk7, cyclin H and MAT1. CAK/cdk7 functions as a master cell cycle regulator by phosphorylating cyclin-dependent kinases for cell cycle progression. We have previously reported that protein kinase C-iota (PKC-iota) associates with CAK/cdk7. In this investigation, immunofluorescence confocal microscopy was used to provide further evidence for the co-localization of PKC-iota with CAK/cdk7. PKC-iota was labeled with Alexa Fluor 488 (green fluorescent dye) and CAK/cdk7 was labeled with Alexa Fluor 555 (red fluorescent dye). The fusion of the red and green fluorescent colors produced a yellow color, which was used to quantify co-localization of PKC-iota and CAK/cdk7. Confocal microscopy revealed the co-localization of PKC-iota with CAK/cdk7 in both the cytoplasm and nucleus of U-373 MG cells.     

A membranotropic region in the C-terminal domain of Hepatitis C virus protein NS4B: Interaction with membranes

We have identified a membrane-active region in the HCV NS4B protein by studying membrane rupture induced by a NS4B-derived peptide library on model membranes. This segment corresponds to one of two previously predicted amphipathic helix and define it as a new membrane association domain. We report the binding and interaction with model membranes of a peptide patterned after this segment, peptide NS4B_H2, and show that NS4B_H2 strongly partitions into phospholipid membranes, interacts with them, and is located in a shallow position in the membrane. Furthermore, changes in the primary sequence cause the disruption of the hydrophobicity along the structure and prevent the resulting peptide from interacting with the membrane. Our results suggest that the region where the NS4B_H2 is located might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex. Our findings therefore identify an important region in the HCV NS4B protein which might be implicated in the HCV life cycle and possibly in the formation of the membranous web.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

Scaffold protein Homer 1: Implications for neurological diseases

Homer proteins are commonly known as scaffold proteins at postsynaptic density. Homer 1 is a widely studied member of the Homer protein family, comprising both synaptic structure and mediating postsynaptic signaling transduction. Both an immediate-early gene encoding a Homer 1 variant and a constitutively expressed Homer 1 variant regulate receptor clustering and trafficking, intracellular calcium homeostasis, and intracellular molecule complex formation. Substantial preclinical investigations have implicated that each of these Homer 1 variants are associated with the etiology of many neurological diseases, such as pain, mental retardation syndromes, Alzheimer's disease, schizophrenia, drug-induced addiction, and traumatic brain injury.     

Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP)

The major hallmark of cellular senescence is an irreversible cell cycle arrest and thus it is a potent tumor suppressor mechanism. Genotoxic insults, e.g. oxidative stress, are important inducers of the senescent phenotype which is characterized by an accumulation of senescence-associated heterochromatic foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). Interestingly, senescent cells secrete pro-inflammatory factors and thus the condition has been called the senescence-associated secretory phenotype (SASP). Emerging data has revealed that NF-κB signaling is the major signaling pathway which stimulates the appearance of SASP. It is known that DNA damage provokes NF-κB signaling via a variety of signaling complexes containing NEMO protein, an NF-κB essential modifier, as well as via the activation of signaling pathways of p38MAPK and RIG-1, retinoic acid inducible gene-1. Genomic instability evoked by cellular stress triggers epigenetic changes, e.g. release of HMGB1 proteins which are also potent enhancers of inflammatory responses. Moreover, environmental stress and chronic inflammation can stimulate p38MAPK and ceramide signaling and induce cellular senescence with pro-inflammatory responses. On the other hand, two cyclin-dependent kinase inhibitors, p16INK4a and p14ARF, are effective inhibitors of NF-κB signaling. We will review in detail the signaling pathways which activate NF-κB signaling and trigger SASP in senescent cells.