Roland Wolkowicz

San Diego State University, San Diego, California, United States

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Publications (13)64.55 Total impact

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    ABSTRACT: The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P < 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation.
    Proceedings of the National Academy of Sciences of the United States of America. 06/2014;
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    ABSTRACT: Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.
    PLoS Pathogens 04/2014; 10(4):e1004045. · 8.14 Impact Factor
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    ABSTRACT: The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 01/2014; 85(1):105-13. · 3.71 Impact Factor
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    ABSTRACT: Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial-targeting sequence (coined "MitoTimer") and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and production was induced with doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Dox addition for as little as 1 h was sufficient to induce MitoTimer expression within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent transition from green to red over 48 h and was amenable to analysis by fluorescence microscopy and flow cytometry of whole cells or isolated mitochondria. A second Dox administration 48 h after the initial induction resulted in a second round of expression of green MitoTimer. The extent of new protein incorporation during a second pulse was increased by administration of a mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.
    Autophagy 10/2013; 9(11). · 12.04 Impact Factor
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    ABSTRACT: Non-peptidomimetic drug-like protease inhibitors have potential for circumventing drug resistance. We developed a much-improved synthetic route to our previously reported inhibitor candidate displaying an unusual quaternized hemi-aminal. This functional group forms from a linear precursor upon passage into physiological media. Seven variants were prepared and tested in cellulo with our HIV-1 fusion-protein technology that result in an eGFP-based fluorescent readout. Three candidates showed inhibition potency above 20μM and toxicity at higher concentrations, making them attractive targets for further refinement. Importantly, our class of original inhibitor candidates is not recognized by two major multidrug resistance pumps, quite in contrast to most clinically applied HIV-1 protease inhibitors.
    Bioorganic & medicinal chemistry 06/2013; · 2.82 Impact Factor
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    ABSTRACT: Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non-host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.
    Proceedings of the National Academy of Sciences 05/2013; · 9.81 Impact Factor
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    ABSTRACT: The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.
    PLoS ONE 01/2013; 8(6):e68835. · 3.53 Impact Factor
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    ABSTRACT: The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral-host interactions or immune activation in their natural milieu.
    Omics: a journal of integrative biology 03/2012; 16(6):312-9. · 2.29 Impact Factor
  • Chitra Rajakuberan, Brett J Hilton, Roland Wolkowicz
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    ABSTRACT: Proteases are essential at different stages of the viral life cycle and for the establishment of a successful infection. Monitoring the catalytic activity of proteases in an easy and straightforward manner can thus drastically facilitate the discovery of novel antivirals, as well as help elucidate the activity and mechanism of action of the viral protease under study. In our laboratory, we have developed an assay in T-cells with a robust read-out to monitor the proteolytic activity of HIV-1 Protease (PR). The assay utilizes the prototypic transcription factor Gal4, which consists of the N-terminal DNA-binding domain and the C-terminal trans-activation domain. The assay is based upon (1) introduction of PR in between the two Gal4 domains to obtain a PR/Gal4 fusion protein and (2) utilization of the enhanced Green Fluorescent Protein as reporter of PR activity.In order to overcome the possible cellular cytotoxicity of PR, the fusion protein in our assay is under the control of a tetracycline-inducible promoter. This ensures that it will be expressed only when needed, upon the addition of tetracycline or doxycycline. When active, PR has autocatalytic activity and cleaves itself from the Gal4 domains, resulting in the inability to induce eGFP expression. However, if PR activity is blocked or it is inactive, the two domains remain intact, resulting in eGFP expression. The assay can therefore be utilized to analyze the inhibitory effects of factors, peptides or compounds, designed on a rational- or nonrational-based approach, in the natural milieu of infection, where eGFP serves as a biosensor for PR activity.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 903:393-405. · 1.29 Impact Factor
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    Brett J Hilton, Roland Wolkowicz
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    ABSTRACT: The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.
    PLoS ONE 01/2010; 5(6):e10940. · 3.53 Impact Factor
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    ABSTRACT: Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery. Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart. In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia-induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia-induced cardiomyocyte death. This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.
    Circulation Research 11/2009; 106(2):307-16. · 11.86 Impact Factor
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    ABSTRACT: To identify positive or negative factors for HIV-1 infectivity, clones from the U937 promonocytic cell line that express similar levels of CD4 and CXCR4, but differ in HIV-1 susceptibility, were compared. In contrast to HIV-1 permissive clone 10 (plus), nonpermissive clone 17 (minus) was adherent to coverslips coated with chemokines, was phagocytic, killed bacteria, and expressed human leukocyte elastase (HLE) in a granule-like compartment (HLEG) that was never detected at the cell surface (HLECS). In contrast to the minus clone, the plus clone expressed HLE on the cell surface and was adherent to coverslips coated with the HLECS ligands alpha1proteinase inhibitor (alpha1PI, alpha1antitrypsin) and the HIV-1 fusion peptide. The phosphorylation status of several important signaling proteins was studied at the single cell level. Tumor suppressor p53, NF-kappaB p65, and Akt were constitutively phosphorylated in the plus clone, but not in the minus clone. Surprisingly, both alpha1PI and LPS induced phosphorylation of NF-kappaB p65 Ser-536 in both clones, but induced dephosphorylation of Ser-529 in the plus clone only. HIV-1 permissivity was conferred to the minus clone in a manner that required stimulation by both alpha1PI and LPS and was coincident to NF-kappaB p65 phosphorylation/dephosphorylation events as well as translocation of HLE to the cell surface. Even when stimulated, the minus clone exhibited greater reverse transcriptase activity, but less p24, than the plus clone. Results presented suggest that HIV-1 uptake and production efficiency are influenced by signaling profiles, receptor distribution, and the phagocytic capacity specific to the stage of differentiation of the CD4+ target cell.
    The Journal of Immunology 02/2008; 180(1):492-9. · 5.52 Impact Factor