SUMO (Small ubiquitin-like modifier) is an important post-translational modification regulating various cellular processes. SUMO modulates the target protein\\'s function and activity by providing an additional noncovalent interaction interface where conjugated SUMO interacts with other proteins. Interaction between SUMO and interacting partner is mediated by the SUMO Interacting Motif (SIM). SIM mediated interaction regulates several cellular processes by changing the SUMOylated protein\\'s intracellular localization, stability, conformation, and enzymatic activity. Viruses modulate cellular pathways to create a suitable environment for survival and replication in the host cell. SUMOylation, being a key regulator of several cellular pathways, is a strategic viral target. Viral proteins exploit both the covalent interaction with SUMO (SUMOylation) and noncovalent SIM-SUMO interactions. Thus, interaction between viral proteins and SUMO, both covalent (SUMOylation) and noncovalent (SIM-SUMO), is important for effective viral infection. The thesis addresses how immediate-early (IE) proteins of Human Cytomegalovirus (HCMV) interact with the host SUMOylation pathway. IE genes are the first viral genes transcribed during the lytic infection cycle. Thus, IEs are essential to optimize the host cell environment for viral genome replication and the transcription of early and late genes. Understanding the molecular details of IE-SUMO interaction can provide us significant insights into how HCMV exploits the host SUMOylation machinery.
HCMV expresses two IE proteins - IE1 and IE2, which are multifunctional and essential proteins for viral propagation. Together IE1 and IE2 help to evade anti-viral responses, modulate the host cell cycle to promote viral genome replication, and transactivate various viral/host promoters. Interestingly, the pathways targeted by IE1 and IE2 (like cell cycle, transcription, immune response) are also regulated by SUMO. Hence, the IEs hijack the host SUMO machinery to control these pathways. The thesis attempts to study the IE1/IE2 interaction with SUMO. We carried out a study to characterize SIMs in HCMV-IE1. Two SIMs were predicted in IE1. NMR confirmed the binding of predicted IE1-SIMs and SUMO. Interestingly, IE1-SIMs did not bind to SUMO2, indicating that IE1-SIMs have paralog specific binding exclusive to SUMO1. The importance of newly identified IE1-SIMs was analyzed for different IE1 functions. IE1-SIM1 facilitates IE1-IE2 synergy to transactivate IE2 responsive promoters. The literature suggests that IE1 mediated transactivation and PML-disruption are co-dependent activities. Our studies showed that the two activities of IE1 are separable. IE1-SIM1 is involved in the IE1-IE2 synergy, but not in IE1 mediated loss of PML SUMOylation. Taken together, the study reveals critical insights into the functional role of noncovalent interactions between E1 and SUMO.
Besides, a systematic study was carried out to study interactions between IE2 and SUMO. NMR titrations confirmed that IE2 has two SIMs. Interestingly, IE2-SIM1 shows a three-fold higher affinity for SUMO1 in comparison to IE1-SIMs. In contrast, IE2-SIM2 binds weakly to SUMO1, but not SUMO2. Interestingly, IE2-SIM1 is adjacent to the IE2 SUMOylation sites. SUMOylation assays and structural studies reveal that IE2-SIM1 recruits E2~SUMO thioester to enhance SUMOylation at K175/K180 up to 4-fold. We revealed two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1. Phosphorylation of the Serines creates new salt- bridges with SUMO residues, which increases the SUMO/IE2-SIM1 affinity by eight-fold. The phosphorylation drastically enhances IE2 SUMOylation as well. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Altogether, we reveal an intriguing mechanism used by the viral factor IE2 to exploit a cross-talk of two post-translational modifications, phosphorylation and SUMOylation, to ensure an effective viral replication.
The prostate is a walnut-sized organ that sits around the male urethra and contributes secretions important for sperm health. With age, the prostate increases in size and constricts the urethral opening. Prostate hyperplasia affects the vast majority of aging men and is a leading cause of urinary problems. Secretions from the prostate drain into the urethra in a region called the transition zone which is the primary site for hyperplasia. I will describe how single cell RNA-sequencing and histological studies led to the identification of two novel cell types, club and hillock, in the urethra and transition zone of the prostate. Unlike prostate cells, these novel urethral cell types do not depend on male hormones for survival. Men with prostate hyperplasia are treated with drugs to reduce the key male hormone dihydrotestosterone and shrink the prostate. Prostate cells in men treated with hormone reducing drugs undergo an unexpected transition to a club cell-like state. This transition is characterized by a reduction in prostate gland size and morphology as well as an increase in NF-KB signaling. This epithelial plasticity appears to be a survival strategy and a protective mechanism in the face of reduced dihydrotestosterone and increased inflammation. The induction of a urethral cell state as a protective mechanism in the prostate highlights understudied aspects of urethral cells in maintaining homeostasis, immune regulation and anti-microbial activity at the urethral barrier.
03:30 PM, Monday, Jul 26, 2021 | Remote VCPresynopsis Thesis Seminar by Ms Aastha Kumari on 'Unique lipid kinase isoforms tune PIP2 resynthesis during G-protein coupled PLC signaling in vivo'