Faculty Bibliography

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Introduction: In addition to its roles as a vascular signaling molecule, nitric oxide (NO) plays roles in metabolism. Mice deficient in eNOS are overweight and develop insulin resistance. It is not known whether the metabolic effects are due to primary roles of NO, or to increased visceral adiposity, leading to secondary metabolic changes. Hypothesis: We hypothesized that NO plays distinct and separable primary roles in white and brown adipogenesis, which underlie the effects on adiposity, energy metabolism, and expression of thermogenic genes. Methods: We exposed wild-type and mice carrying specific gain of function and loss of function eNOS mutations to cold at 4C for 48 hours and assessed expression of thermogenic gene programs in white and brown adipose tissue. To study cell autonomous effects, we differentiated adipocyte precursors from brown and white fat in the presence of NOS inhibitors and NO donors, as well as with siRNA to knockdown eNOS expression. Results: Cold exposure resulted in upregulation of the thermogenic gene program in brown adipose tissue. Animals carrying a gain of function mutation in eNOS showed increased UCP1 expression even without cold exposure. Induction of thermogenic genes was more pronounced in the animals with gain of function eNOS mutation. Differentiation of adipocyte precursors showed effects of eNOS on adipogenesis. Cells treated with the pharmacologic blockade (L-NAME and L-NA) as well as genetic knockdown (siRNA) showed dose-dependent inhibition of adipocyte differentiation. MitoTracker Red CMXRos staining showed that treatment with the NO donor SNAP increases mitochondrial biogenesis, while L-NAME decreases mitochondrial biogenesis. Conclusions: We show that eNOS-derived NO plays distinct and separable roles in white and brown adipogenesis. In brown adipocytes, eNOS regulates the expression of the thermogenic gene program, with upregulation of expression even without cold exposure, and greater increase in response to cold. In white adipocytes, eNOS-derived NO is required for adipocyte differentiation and mitochondrial biogenesis

Obesity is a complex multifactorial disease. Adipocytes arise from pluripotent mesenchymal stem cells (MSC), which are also capable of differentiation into bone, muscle, or cartilage. Adipogenesis involves lineage commitment, mitotic clonal expansion, and terminal differentiation. Understanding these mechanisms, as well as when and how to turn them on or off, may allow development of new therapeutic approaches to obesity, diabetes, and cardiovascular disease. The most abundant non-lipid component of olive plant is the polyphenol oleuropein (Ole). We found that Ole modulates adipocyte differentiation, fat accumulation and adipogenic gene expression in human MSCs (hMSC). Ole blocks adipogenesis in a dose-dependent manner. Using RT-PCR to monitor gene expression, we found that Ole down-regulates the expression of adipogenic genes PPAR-gamma-2, LPL (lipoprotein lipase), and aP2 (lipid binding protein), while it upregulates PPAR-delta expression. In addition, in the presence of Ole, we were able to achieve transdifferentiation and dedifferentiation, allowing fat cells to assume other fates. These results demonstrate the potential utility of Ole for the treatment of obesity, diabetes, and related disorders, which are associated with increased fat mass. Because it modulates adipocyte differentaion, Ole may also be useful for the treatment of cachexia and lipodystrophy

We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection

HL9 is a nonapeptide fragment of human lysozyme which has been shown to have anti-HIV-1 activity in nanomolar concentration. This study aims to explain this inhibitory activity by using molecular dynamics (MD) simulation, focusing on the ectodomain of gp41, the envelope glycoprotein of HIV-1 crucial to membrane fusion. It was found that in HL9, two Trp residues separated by two others occupy the conserved hydrophobic pocket on gp41 and thus inhibit fusion in dominant-negative manner. Detailed HL9-gp41 binding interactions and free energies of binding were obtained through MD simulation and solvated interaction energies (SIE) calculation, giving a binding free energy of -8.25 kcal/mol which is in close agreement with the experimental value of -9.96 kcal/mol. Since C-helical region (C34) of gp41 also has two Trp residues separated by two others, this arrangement may be generalised and used to scan peptide library and to find those having similar manner of inhibition

A series of norbornane-based HIV-1 protease (PR) inhibitors are designed theoretically to displace the tetrahedrally coordinated internal water molecule that bridges inhibitor to flaps via hydrogen bonds. These designed inhibitors use the norbornenone oxygen atom to mimic this structural water molecule and contain diols to interact with the carboxylate oxygens of catalytic aspartates. The binding free energies were estimated by modified linear interaction energy approach [Zoete H, Michielin O, Karplus M, J Comput Aided Mol Des 17: 861, 2003], in which the binding free energy is written as a linear combination of the electrostatic interaction energy between PR and the ligand, E-elec, the van der Waals interaction energy between PR and the ligand, E-vdW, and the difference of the solvation free energies of the complex, the receptor, and the isolated ligand, Delta G(solv). The equation obtained in previous work [Da W. Zhang, Philip Lin Huang, Sylvia Lee-Huang, John Z. H. Zhang, J Theor Comput Chem 7:485, 2008] is applied directly to calculate the binding free energy of designed norbornane-based HIV-1 PR inhibitors