The mitochondrial unfolded protein response (UPR^mt) is a key cellular mechanism for maintaining mitochondrial function in the face of oxidative and proteotoxic stress. This pathway upregulates mitochondrial chaperones and proteases that aid in folding and degradation of aberrant proteins. Induction of the UPR^mt was observed to correlate with increased longevity in the nematode worm Caenorhabditis elegans, a model whose genetic system is widely used to study the molecular mechanisms of aging. Through a genome-wide RNAi screen for regulators of the UPR^mt, we found that deficiency in the transaldolase (tald-1) resulted in activation of the UPR^mt. TALD-1 is responsible for the production of NADPH in the oxidative phase of the pentose phosphate pathway (PPP). In subsequent studies, we have found that knockdown of transaldolase reduces in vivo mitochondrial respiration, changes the overall metabolism of lipids, and affects the activity of transcription factors that regulate a starvation response and expression of metabolic genes. Among the genes transcriptionally altered by transaldolase knockdown is the flavin-containing monooxygenase-2 (fmo-2) gene, which has been previously found to be regulated by starvation. Like starvation, tald-1(RNAi) induced nuclear localization of HLH-30, a transcription factor responsible for the expression of autophagy genes. HLH-30 is also required for fmo-2 up-regulation. Epistasis lifespan analyses using tald-1 (RNAi) and deletion mutant strains of C. elegans, quantitative gene expression studies, and fluorescence reporter assays were used to determine the mechanistic relationship between tald-1, hlh-30, and fmo-2 as regulators of longevity in C. elegans.

As the primary producers of energy (ATP) in a cell, the mitochondria are key players in controlling cell aging and death. Interestingly, mild oxidative stress, derived from mitochondrial dysfunction or exogenous agents, evokes adaptive stress response pathways that can increase healthspan and lifespan in the nematode worm Caenorhabditis elegans. One of the pathways that responds to mitochondrial stress is the mitochondrial unfolded protein response (UPR^mt). The UPR^mt responds to stress from unfolded proteins or oxidative damage by upregulating expression of chaperones that stabilize and assist folding of unfolded proteins as well as proteases that can degrade proteins within the mitochondria. To search for genes that regulate the UPR^mt, we performed a genome-wide RNAi screen and found that knockdown of the tald-1 gene activates the UPR^mt. The tald-1 gene encodes transaldolase, an enzyme in the pentose phosphate pathway (PPP). One role of the PPP is to generate NADPH, which is required for cellular redox homeostasis. Although transaldolase deficiency has been linked to mitochondrial dysfunction in humans and mice, how the PPP controls mitochondrial function is not well understood. The focus of our study was to determine the relationship between PPP inhibition, mitochondrial function, and longevity by investigating the role of transaldolase in redox homeostasis and regulation of redox sensitive signaling pathways. Here we report that transaldolase deficient worms induced a starvation-like metabolic response that requires JNK MAPK signaling, a pathway that is directly activated by oxidative stress, to increase lifespan. Since the UPR^mt and PPP are correlated with several age-related diseases such as cancer and neurodegenerative disease, understanding the underlying genetics and molecular mechanisms of these pathways and their role in aging is likely to be relevant for human health.
 

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS)—the most common malignancy of AIDS patients worldwide. Because KS is an endothelial cell based cancer, it relies on mechanisms unique to endothelial cells to proliferate. One such way is through expression of vascular endothelial growth factor receptors 1 and 3 (R1 and R3). The goal of my project is to determine the viral mechanisms by which KSHV upregulates these receptors. Because the majority of KSHV-infected cells are in latent phase, I hypothesized that latent genes are responsible for R1 and R3 upregulation. By infecting cells with an adenovirus modified to express only genes within KSHV latency-associated region (KLAR), I was able to confirm that KLAR genes are sufficient to upregulate R1, but not R3. To determine which KLAR genes are required for R1 upregulation, I created lentiviruses to express one viral gene at a time. By infecting cells with these lentiviruses and monitoring R1 mRNA transcript production by PCR, I was able to rule out three of the four genes in KLAR. Using a cell line modified to constituently express the remaining KLAR gene, latency-associated nuclear antigen (LANA), I found mRNA transcript levels for R1 were significantly higher in LANA-expressing cells than in control cells. This suggests LANA is responsible for R1 upregulation. I am currently conducting a luciferase assay to determine if LANA interacts with the R1 promoter to modify R1 expression. To narrow the search for genes involved in R3 upregulation, I will continue to make lentiviruses to test the involvement of viral genes not located in KLAR. I will present current work examining if conditioned media induces R3 to determine if virus induced secreted factors could be responsible for R3 upregulation. My research will contribute to the ongoing search for treatment strategies for KSHV and KS.

A more complete understanding of a protective HIV-specific immune response is critical to designing effective vaccine strategies. Researchers have characterized hundreds of antibodies (Abs) that elicit broad and potent neutralizing activity but much less is known about Abs that mediate FcR-dependent responses, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular virus inhibition (ADCVI) activity. ADCC involves antibody-dependent recruitment of natural killer cells through Fc/FcR-dependent interactions and subsequent lysis of infected cells whereas ADCVI activity includes ADCC but also involves chemokine release and FcR-mediated phagocytosis. Intriguing evidence from the recent RV144 vaccine trial demonstrated a modest 31.2% protection that was subsequently correlated with higher levels of ADCC activity, rendering previously uncharacterized ADCC- and ADCVI-mediating Abs of increased interest. Here we describe 13 HIV-specific Abs identified from an individual who developed a uniquely broad and potent HIV-specific response early following infection. All 13 Abs were tested for neutralizing and ADCC activity and a small subset were tested for ADCVI activity. While only two Abs demonstrated modest neutralizing activity, two Abs demonstrated broad, cross-clade ADCC activity as well as ADCVI activity. Our project aims to firstly evaluate all 13 HIV-specific Abs for ADCVI activity and secondly, to map the binding properties of the mAbs identified to mediate ADCVI. These latter studies will be completed first using a high-throughput binding antibody multiplex assay (BAMA), and secondly through a more refined series of competition-based enzyme-linked immunosorbent assays (ELISAs). Preliminary analysis has indicated that at least one of these 13 Abs mediates ADCVI but neither neutralizing nor ADCC activity and mapping studies suggest this Ab may bind to the gp41 portion of the HIV envelope. Further understanding of the epitope specificities of Abs that mediate ADCVI activity will help us characterize how this individual mounted a broad and potent response early following infection.

Human genetic variants—genetic differences between people—are common. The functional significance of these variants is often not known, hence they are frequently referred to as variants of unknown significance (VUS). We have focused on developing experimental approaches to determine the functional significance of VUS, as in silico approaches have low predictive power. Our genes of choice are mutated in Fanconi anemia (FA), a rare recessive human disease that leads to early bone marrow failure and a risk of leukemia followed by a high lifetime risk of head and neck cancer. We have focused further on the two FANC genes that are mutated in 2/3 of FA patients, FANCA and FANCG. In order to functionally phenotype all small genetic variants in these two genes, we are first generating a plasmid library of 593 different mutant alleles, each linked to a unique barcode sequence. These plasmids are then used to generate a corresponding library of isogenic U-2 OS cells that each expresses a single FANCA or FANCG variant protein. Our functional assay is to determine how well each variant protein supports cell survival, after depletion of the corresponding endogenous FANC protein, upon treatment with increasing doses of the DNA cross-linking drug MMC. Mutant survival or fitness of specific alleles as a function of MMC dose is determined by barcode sequencing and counting to compare specific variant allele loss or persistence versus wildtype and dead mutant protein controls. My presentation will include assay development, validation results, and phenotyping data on a small number of FANCG genetic variants. This approach and methods are general, and should be applicable for determining the functional phenotype of VUS in any gene for which there is a good cell-based functional assay. 

Collateral artery development is the body's natural adaptive response to bypass major artery blockage. However, this process restores about 30% of the original blood flow, which is insufficient to avoid end-organ ischemia. Therefore, there is a great need for better understanding of the angiogenesis process. Overexpression of the p27kip1 (p27 or cdkn1b) gene inhibits angiogenesis in mice, and p27 knock-out mice (p27-/- or KO) have improved angiogenesis and arteriogenesis. We previously showed that p27-/- mouse vascular smooth muscle cells (VSMC) have increased matrix metalloproteinase 2 (MMP2) mRNA and migrate more effectively than wild type (WT) VSMC. We wanted to better understand the link between p27, MMP2, and cell repair capability. We hypothesized that decreasing the p27 genetic dose would result in increased MMP2 expression. Experiments were performed on p27+/+ (WT), p27+/- (HET), and KO VSMC isolated from 8-10 wk old female mouse aortae. All groups were tested for MMP2 mRNA and protein expression level using qRT-PCR and Western blot respectively. A cytoplasmic fraction was used for protein analysis. MMP2 mRNA expression in HET VSMC was significantly higher than in WT (194±9 vs. 101±3%, p<0.001), and significantly lower than in KO VSMC (280±38%, p<0.001). TIMP1 and TIMP2 mRNA expression was significantly lower in the KO VSMC than in the HET and WT cells (p<0.01). In the cytoplasmic fraction there was no significant difference between MMP2 protein levels between KO and HET VSMC, but both were significantly higher than the level in WT (2.55±0.27 vs. 2.15±0.22 and 1.14±0.3 respectively, p<0.05). Decreasing genetic dose of p27 in VSMC leads to higher levels of MMP2 mRNA and protein levels. This suggests that p27’s effect on VSMC migration in vitro and possibly its effect on angiogenesis and arteriogenesis is through regulation of MMP2 expression. TIMP1 and TIMP2 may also play a role in this process.

Kaposi’s sarcoma (KS) is the most common tumor found in sub-Saharan Africa and in AIDS patients worldwide. It is a highly angiogenic tumor caused by Kaposi’s sarcoma-associated herpesvirus (KSHV), which establishes a latent infection in endothelial cells. KSHV infected endothelial cells display many angiogenic phenotypes, including vascular tube formation and stabilization. Recent data shows a link between KSHV angiogenesis and the hypoxia inducible factors (HIF) pathway. HIFs are a family of heterodimeric transcription factors that respond to low levels of oxygen in the cell. The HIF pathway has been shown to activate pro-angiogenic genes, including vascular endothelial growth factor (VEGF). Our lab has previously shown that both HIF-1alpha and HIF-2alpha are upregulated during KSHV infection. Although HIF-1alpha is expressed ubiquitously, HIF-2alpha is specific to endothelial cells. I hypothesize that HIF-2alpha regulates angiogenesis during KSHV infection. To test this hypothesis, I knocked down HIF-2alpha expression in endothelial cells using an shRNA lentivirus, which successfully knocked down expression by 80%. I then conducted a capillary tube assay on mock and KSHV infected cells. This assay involves homogenous seeding of cells onto Matrigel, a basement membrane matrix for studying angiogenesis in vitro. The cells attach and migrate on the Matrigel to organize into capillary-like structures, which mimics the later stages of angiogenesis. Latent KSHV infection has been shown to form tubes at 6 hours post-plating in Matrigel and to stabilize tube formation at 24 hours, a time point when capillary-like formation normally regresses in uninfected cells. My preliminary data shows that when HIF-2alpha is knocked down, KSHV-induced tube formation is inhibited. Future work will explore the viral mechanism and the cellular significance of HIF-2alpha upregulation during latent KSHV infection. If HIF-2alpha is required for KSHV-induced angiogenesis, this transcription factor may be a promising therapeutic target for KS tumors.

Transient receptor potential vanilloid receptor 1(TRPV1) is a nonselective cation channel whose activity can be regulated by different kinds of factors including vanilloids such as capsaicin. Nerve growth factor (NGF) has been shown to cause the rapid sensitization of nociceptors neurons expressing TRPV1 under inflammatory hyperalgesia. In other words, TRPV1 is involved the sensation of pain caused by inflammation. It’s been suggested that tropomyosin receptor kinase A (trkA) and its downstream effectors including phosphoinositide 3-kinase (PI3K) play a key role in TRPV1 potentiation and we want to further examine the molecular signaling mechanism. For the purpose of our study, we utilize a system based on a light-sensitive molecule phytochrome B (PhyB) to mimic inflammatory responses in cells. Under 650nm light, PhyB undergoes conformational change and binds to phytochrome interaction factor (PIF). By fusing proteins of interest to PIF, PhyB-PIF interaction controlled by light can be used to manipulate signaling pathways including PI3K pathway. To study the potentiating effect of PI3K on TRPV1, we examine NIH3T3 cells transfected with proteins of interest using calcium imaging. We compare cell responses to capsaicin before and after PI3K translocation activated by 650 nm light. Our experimental data suggest a significant increase of calcium signalling after translocating PI3K to the plasma membrane. In the future, we are hoping to learn more about how the downstream effectors can affect TRPV1 trafficking. We will perform similar procedures as described using dominant-negative Akt and Rac1 cells and investigate the specific mechanisms. Eventually, we hope to utilize what we know about TRPV1 in pain treatment development.