Funded Projects

*New* RFA applications are currently open! .
The last day for all submissions is Monday, February 3rd @ 1:00 am

The relative ability of cell autonomous HIV-1 restriction factors to interfere with the viral life cycle contributes to a host’s level of susceptibility to infection. Pharmacological enhancement of restriction factor efficacy would be a novel approach to treating HIV infection. However, the mechanistic basis for HIV blockage by restriction factors is not completely understood hampering efforts to employ restriction factor-based host directed therapies. The tripartite motif (TRIM) family of proteins consists of more than 70 members in humans, several of which have been identified as antiviral restriction factors. In this role, TRIMs can diminish viral replication directly by interfering with the viral life cycle or indirectly by fine tuning cellular innate immune responses. TRIM family member TRIM5α accomplishes both of these: first, it prevents retroviral infection of cells by a hitherto unexplained mechanism. Second, TRIM5α also acts as a pattern recognition receptor, promoting the establishment of an antiviral cellular state via the activation of inflammatory signaling pathways upon retroviral recognition. Although TRIMs appear to employ multiple approaches in antiretroviral defense, one strikingly common feature among the TRIM family is that many if not all TRIMs are involved in the regulation and execution of autophagy. In addition to its role as a known defense mechanism against intracellular pathogens (including HIV-1), autophagy is also increasingly recognized as a means of reducing or fine tuning inflammation. Here, we propose to test the hypothesis that autophagy underlies TRIM action in protecting cells against HIV-1 infection and in modulating the TRIM-dependent inflammatory signaling. The studies proposed here have several overarching goals. First, they seek to improve our understanding of the molecular mechanism whereby rhesus TRIM5α both regulates autophagy and directs the autophagic degradation of incoming HIV-1 capsids (Aim 1). Second, they will determine if modulations of the autophagy pathway affect TRIM5α-dependent activation of pro-inflammatory signaling upon lentiviral infection. Finally, they will address whether human TRIMs other than TRIM5α that restrict HIV also employ autophagy in their antiviral actions (Aim 2). We have assembled a team of autophagy and HIV experts to address these questions. Our studies have the potential to uncover the mode of action of several known antiretroviral proteins and lay the groundwork for our understanding of how TRIMs as a family can both positively and negatively affect inflammation. We expect these studies to show that autophagy is a unifying aspect of diverse TRIM actions in HIV defense. Since autophagy can be pharmacologically manipulated, our findings may indicate that modulations of autophagy could be a therapeutic approach to dealing with TRIM-related diseases including HIV/AIDS. Our expertise in TRIMs and autophagy, along with the financial and institutional support to be provided should the COBRE application be funded will ensure successful completion of these aims.

Members of the TRIM family of proteins have been identified as being capable of limiting the ability of HIV to infect and replicate within cells, but the mechanism(s) underlying their action have not been fully uncovered. In this proposal, we test the hypothesis that autophagy, a pathway that can be manipulated with existing drugs, underlies the action of TRIM proteins in antiviral defense. These studies will further our understanding of the cellular functions of TRIM proteins at a molecular level and may provide the groundwork for therapeutic approaches to the diverse diseases, including HIV/AIDS, in which TRIMs play a role.

NIH

The function of T helper (TH) cells, the central organizers of adaptive immunity, is specified by the effector cytokines they produce. Regulation of TH cell cytokine secretion is not well understood and represents an important gap in our knowledge. Our recent data indicate that membrane- associated nucleic acid binding protein (Mnab, encoded by Rc3h2) is required for TH cell effector cytokine secretion. Mnab shares with its paralog Roquin (encoded by Rc3h1) a highly conserved N-terminus but possesses a unique hydrophobic C-terminus. Roquin is important for control of follicular helper T (Tfh) cell development through repression of Icos mRNA. Recently, Mnab was shown to play a redundant role with Roquin in Tfh cell development via repression of Icos and Ox40 mRNAs. Whether Mnab also targets other mRNAs and regulates function of other TH lineages is unclear. Our preliminary studies demonstrated that a Mnab deficiency led to profound defects in effector cytokine production in TH1, TH2 and TH17 cells, which differs from its known function, suggesting an important role of the distinct C-terminus of Mnab. Based on our preliminary data, we formulate a novel hypothesis: Mnab targets mRNAs encoding proteins in the stress response-autophagy pathway, a common pathway that is critical in TH cell effector cytokine production. The overall specific aims of this project are: Aim 1. Determine the role of Mnab and UPR-autophagy in TH cell function. Aim 2. Delineate the molecular mechanism whereby Mnab controls mRNA stability. Aim 3. Determine the role of Mnab in TH cell function in vivo using disease models. By addressing our hypothesis, these studies will reveal a novel post-transcription control mechanism of TH cell effector function. Manipulation of the corresponding pathways may be of therapeutic benefit in human disease, such as autoimmune and inflammatory disorders.

The function of T helper (TH) cells, the central organizers of adaptive immunity, is specified by the effector cytokines they produce. Regulation of TH cell cytokine secretion is not well understood and represents an important gap in our knowledge. In our preliminary studies, we found that in vitro, deficiency of membrane-associated nucleic acid binding protein (Mnab) led to profound defects in TH cell effector cytokine production. How Mnab regulates TH cell effector function is entirely unclear. We hypothesize that Mnab modulates stability of mRNAs encoding proteins in a common pathway that is critical in TH cell cytokine production. Our current data support a novel hypothesis that Mnab stabilizes mRNAs in the stress response-autophagy pathway that is required for secretory cells. In the proposed study, we will further understand how Mnab controls TH cell cytokine secretion and whether Mnab regulates TH cell function in disease models. Our studies will reveal a novel post-transcriptional control mechanism of TH cell effector function. Manipulation of the corresponding pathways may be of therapeutic benefit in human disease, such as autoimmune and inflammatory disorders.

NIH

Graduated Projects

Brown and Beige (or brite) adipose tissues burn lipid by converting chemical energy into heat, and have been considered as a new therapeutic target to counteract obesity. The regulation of thermogenic function in adipose tissue at the transcriptional level has been extensively studied in the past several years. However, the upstream signaling pathways that control the transcriptional machinery of thermogenic genes and effector mechanisms in brown or beige adipocytes remain largely unknown. Our recent study demonstrated that overactivation of mTOR Complex 1 (mTORC1) signaling is associated with impaired thermogenic function in vivo (Liu et al., 2014, Cell Metabolism). Consistent with this, our preliminary data showed that inactivation of mTORC1 by adipose-specific ablation of raptor, a key component of mTORC1, up-regulated the expression of thermogenic genes in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT or beige fat). In addition, we also found that the autophagy, a pathway downstream of mTORC1 is activated by cold stress and a β3-adrenoceptor agonist in vivo and in cells, and inhibition of autophagy diminishes β3-adrenoceptor agonist- induced UCP1 expression in primary adipocytes. In support of this, inhibition of autophagy by adipose-specific deletion of autophagy protein 7 (ATG7) suppresses basal and cold-induced energy expenditure, lipolysis and UCP1 expression in iWAT in vivo. Based on these findings, we hypothesize that autophagy plays a critical role in regulating the browning of white adipose tissue and mediates the beneficial effect of mTORC1 inhibition on thermogenesis in human brown adipocytes. We will first determine whether and how inhibiting mTORC1 and autophagy alters beige adipocyte differentiation and thermogenesis via cell-autonomous mechanisms. We will then investigate whether autophagy is an essential effector downstream of mTORC1 in regulating thermogenesis in WAT using adipose-specific autophagy-related protein 7 (ATG7)/raptor double KO mice. Lastly, we will delineate the role of mTORC1 and autophagy in regulating thermogenesis in primary human brown adipocytes. This study will lead to the identification of the mTORC1/autophagy pathway as a critical regulator of beige adipocyte differentiation and recruitment in response to various environmental factors. In addition, elucidation of the underlying signaling mechanisms involved in the browning of white fat may reveal promising new anti-obesity drug targets and lead to novel therapeutic approaches for obesity-associated metabolic diseases.

Brown and brown‐like (beige or brite) adipocytes in adipose tissue have strong anti‐obesity and anti‐ diabetic benefits. However, the mechanisms underlying the browning of white adipose tissue remains largely unknown. This proposed study investigates how mTORC1 and autophagy pathways cooperate to regulate the recruitment and activation of beige adipocytes in white adipose tissue. The results from this study may lead to the identification of potential therapeutic targets for the treatment of obesity and its associated metabolic diseases.
Received an independent R01; Completed October 2018

NIH

The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and inflammatory bowel disease (IBD). Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. The overall specific aims of this application are: Specific Aim 1. To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier. Specific Aim 2. To elucidate the role of intracellular signaling in autophagy regulation of intestinal TJ barrier. Specific Aim 3. To delineate the role of autophagy in intestinal TJ barrier function and inflammation in animal models of IBD. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.

The tight junctions (TJ) present between intestinal epithelial cells act as a paracellular barrier and serve as a first line of defense against permeation of noxious antigens present in the intestinal lumen. Defective intestinal tight junction (TJ) barrier allows penetration of harmful luminal antigens in the gut which in turn leads to intestinal inflammation. Autophagy is a normal process that helps cell survival by recycling the nutrients and energy via degradation and turnover of the misfolded or unnecessary proteins. Recent studies have shown that mutations in autophagy related genes and defects in autophagy process are risk factors for inflammatory bowel disease (IBD) including Crohn disease (CD). The purpose of this grant application is to elucidate the mechanisms involved in autophagy-mediated enhancement of the intestinal epithelial tight junction barrier. Clinically, maintenance of mucosal barrier in the intestine is critical for the prevention of intestinal mucosal damage and therapeutic success in IBD cases. This study will provide novel insights into the crucial role of autophagy in enhancement of intestinal barrier and prevention of intestinal inflammation.
Received an independent R01; Completed January 2018

NIH