Browsing by Author "Jambunathan, Nithya"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Caveolin-1 knockout mitigates breast cancer metastasis to the lungs via integrin α3 dysregulation in 4T1-induced syngeneic breast cancer model
    (Springer Nature, 2024) Singh, Dhirendra Pratap; Pathak, Rashmi; Chintalaramulu, Naveen; Pandit, Abhishek; Kumar, Avinash; Ebenezer, Philip J.; Kumar, Sanjay; Duplooy, Alexander; White, Mary Evelyn; Jambunathan, Nithya; Dharmakumar, Rohan; Francis, Joseph; Radiology and Imaging Sciences, School of Medicine
    Caveolin-1 (Cav-1) is a critical lipid raft protein playing dual roles as both a tumor suppressor and promoter. While its role in tumorigenesis, progression, and metastasis has been recognized, the explicit contribution of Cav-1 to the onset of lung metastasis from primary breast malignancies remains unclear. Here, we present the first evidence that Cav-1 knockout in mammary epithelial cells significantly reduces lung metastasis in syngeneic breast cancer mouse models. In vitro, Cav-1 knockout in 4T1 cells suppressed extracellular vesicle secretion, cellular motility, and MMP secretion compared to controls. Complementing this, in vivo analyses demonstrated a marked reduction in lung metastatic foci in mice injected with Cav-1 knockout 4T1 cells as compared to wild-type cells, which was further corroborated by mRNA profiling of the primary tumor. We identified 21 epithelial cell migration genes exhibiting varied expression in tumors derived from Cav-1 knockout and wild-type 4T1 cells. Correlation analysis and immunoblotting further revealed that Cav-1 might regulate metastasis via integrin α3 (ITGα3). In silico protein docking predicted an interaction between Cav-1 and ITGα3, which was confirmed by co-immunoprecipitation. Furthermore, Cav-1 and ITGα3 knockdown corroborated its role in metastasis in the cell migration assay.
  • Thumbnail Image
    Item
    Inactivation of the UL37 Deamidase Enhances Virus Replication and Spread of the HSV-1(VC2) Oncolytic Vaccine Strain and Secretion of GM-CSF
    (MDPI, 2023-01-27) Clark, Carolyn M.; Jambunathan, Nithya; Collantes, Therese M. A.; Kousoulas, Konstantin G.; Medicine, School of Medicine
    The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.
  • Thumbnail Image
    Item
    Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein
    (MDPI, 2022-10-04) Collantes, Therese Marie A.; Clark, Carolyn M.; Musarrat, Farhana; Jambunathan, Nithya; Jois, Seetharama; Kousoulas, Konstantin G.; Medicine, School of Medicine
    The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.