Avelino Teixeira

Prothymosin-α inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8+ T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8+ cells. Thus, a protein produced by CD8+ T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTα may provide therapeutic leads for IFN-sensitive viruses.

Novel Function of Prothymosin Alpha as a Potent Inhibitor of Human Immunodeficiency Virus Type 1 Gene Expression in Primary Macrophages

ABSTRACT CD8+ T lymphocytes control human immunodeficiency virus type 1 (HIV-1) infection by a cytotoxic major histocompatibility complex-restricted pathway as well as by secretion of noncytotoxic soluble inhibitory factors. Several components of CD8+ cell supernatants have been identified that contribute to the latter activity. In this study we report that prothymosin alpha (ProTα), a protein found in the cell culture medium of the herpesvirus saimiri-transformed CD8+ T-cell line, K#1 50K, has potent HIV-1-inhibitory activity. Depletion of native ProTα from an HIV-1-inhibitory fraction of CD8+ cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTα is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. In this report we demonstrate that ProTα suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTα protein potently inhibit HIV-1 long terminal repeat (LTR)-driven gene expression in macrophages. Furthermore studies using different promoters in lentiviral vectors (cytomegalovirus and phosphoglycerate kinase) revealed that suppression of viral replication by ProTα is not HIV LTR specific.

Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel

We have described previously a cell surface channel that is highly selective for nucleic acids. Nucleic acid conductance is 10 pS and the channel is at least 10,000-fold more selective for oligodeoxynucleotides than any anion tested (1). Herein we provide evidence that the nucleic acid-conducting channel (NACh) is a heteromultimeric complex of at least two proteins; a 45-kDa pore-forming subunit (p45) and a 36-kDa regulatory subunit (p36). Reconstitution of p45 in planar lipid bilayers resulted in formation of a channel which gated in the absence of nucleic acid and which was more selective for anions (including oligonucleotide) than cations. This channel exhibited transitions from one level of current to another (or to the closed state); however the incidence of transitions was rare. Channel activity was not observed when p36 was reconstituted alone. Reconstitution of p36 with p45 restored nucleic acid dependence and selectivity to the channel. Protein sequence analysis identified p36 as cytosolic malate dehydrogenase (cMDH). Experiments were performed to prove that cMDH is a regulatory subunit of NACh. Selective activity was observed when p45 was reconstituted with pig heart cMDH but not with mitochondrial MDH. Both the enzyme substrate l-malate and antiserum raised against cMDH block NACh activity. These data demonstrate that a nucleic acid conducting channel is a complex of at least two proteins, p45 and cMDH. Furthermore, these data establish that cMDH confers nucleic acid selectivity of the channel.

Thyroid and Bone: Macrophage-Derived TSH-β Splice Variant Increases Murine Osteoblastogenesis

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-β subunit, designated TSH-βv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-βv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-β, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Influence of Prothymosin-α on HIV-1 Target Cells

Abstract:  The important role of CD8+ T cells in controlling HIV‐1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)‐dependent cytotoxic activity of CD8+ T cells, they produce soluble factors that suppress HIV‐1 replication in an MHC‐independent manner. Several of those factors have been identified, including β‐chemokines, Rantes, MIP‐1α, MIP‐1β, and MDC. We previously identified that prothymosin alpha (ProTα) in the conditioned medium of HVS transformed CD8+ T cells was a potent inhibitor of HIV‐1 replication following proviral integration. In this report we further characterize the anti‐HIV‐1 activity of ProTα by demonstrating its target‐cell specificity, distinction from additional inhibitors of HIV‐1 transcription in CD8+ T cell supernatants, as well as the differential regulation of host cell antiviral genes that could impact HIV‐1 replication. These genes include a number of transcription factors as well IFN‐α‐inducible genes including PKR, IRF1, and Rantes, in the absence of induction of IFN‐α. These data suggest that the anti‐HIV‐1 activity of ProTα is mediated through the modulation of a number of genes that have been reported to suppress HIV‐1 replication including the dysregulation of transcription factors and the induction of PKR and Rantes mRNA.

Effect of target cell availability on HIV-1 production in vitro

Objective The recovery of CD4 target cells following antiretroviral therapy may facilitate virus production and escape from antiretroviral suppression. To address this hypothesis, we directly examined whether the CD4 target cell number increases viral production in the presence of suboptimal therapy. Design The effect of the CD4 T cell number on HIV-1 replication with a suboptimal dose of zidovudine was studied in vitro. Methods Varying numbers of CD4 T cells were infected with HIV-1 and treated with 1 nM zidovudine. Virus production was measured by p24 antigen capture enzyme-linked immunosorbent assay. Partial sequencing of HIV-1 pol was performed to assess zidovudine-resistant mutations. Results Wild type virus production was found to increase eightfold in cultures with 100 × 104 cells compared with cultures with 10 × 104 cells. The IC90 of zidovudine was 4 logs higher in cultures with 16 × 104 cells compared with cultures with 1 × 104 cells. No zidovudine-resistant mutations were found. Conclusion Target cell availability may play a direct role in wild type HIV-1 resurgence following therapy.

Proteomics analysis of the non-muscle myosin heavy chain IIa-enriched actin-myosin complex reveals multiple functions within the podocyte.

MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA‘s function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA’s functions goes beyond the podocyte, providing important insights into its general molecular role.

Multiple Breast Cancer Cell-Lines Derived from a Single Tumor Differ in Their Molecular Characteristics and Tumorigenic Potential

Background Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient’s breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. Methods Five breast cancer cell lines were derived from a single patient’s primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fuorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. Results We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. Conclusions All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer.

Prothymosin α Variants Isolated From CD8+ T Cells and Cervicovaginal Fluid Suppress HIV-1 Replication Through Type I Interferon Induction

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.

NACh is a nucleic acid–conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK1 cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or l-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK1 cells. LLC-PK1 cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK1 cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.