The ESCRT apparatus has multiple Ubiquitin-binding domains and participates in a wide variety of cellular processes. Many of these ESCRT-dependent processes are keenly regulated by Ub, which serves as a lysosomal sorting signal for membrane proteins targeted into multivesicular bodies and which may serve as a mediator of viral budding from the cell surface. Hints that both ESCRTs and Ub work together in the processes such as cytokinesis, transcription, and autophagy are beginning to emerge. Here we explore the relationship between ESCRTs and Ub in MVB sorting and viral budding.

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic HECT domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly, and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function, and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 in vivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface, and are less active when they are "unlocked" by Ubp2mediated deubiquitination.

A chimeric Cu-binding peptide has been designed on the basis of a turn substitution of the prion (PrP) octarepeat Cu-binding site into the engrailed homeodomain helix-turn-helix motif (HTH). This system is a model for the investigation of a single PrP Cu-binding site in a defined protein context. The 28-mer Cu-HTH peptide P7 spectroscopically mimics the PrP octarepeat (P7 = TERRRQQLSHGGGWGEAQIKIWFQNKRA). The Cu(II)-binding affinity of P7 was determined by ESI-MS and tryptophan fluorescence titrations to be K(d) = 2.5 +/- 0.7 microM at pH = 7.0. The quenching of fluorescence of the Trp within the binding loop (underlined above) is pH dependent and highly specific for Cu(II). No Trp quenching was observed in the presence of divalent Zn, Mn, Co, Ni, or Ca ions, and ESI-MS titrations confirmed that these divalent ions do not appreciably bind to P7. The EPR spectrum of Cu(II)-P7 shows that the Cu environment is axial and consistent with 6-coordinate N(3)O(H(2)O)(2) or N(4)(H(2)O)(2) coordination (A( parallel) = 172 x10(-)(4) cm(-)(1); g( parallel) = 2.27), very similar to that of the PrP octarepeat itself. Also like PrP, circular dichroism studies show that apo P7 is predominantly disordered in solution, and the structure is slightly enhanced by Cu binding. These data show the Cu-PrP HTH peptide reproduces the Cu-binding behavior of a single PrP octarepeat in a new context.

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Background: The COVID-19 pandemic has affected billions of lives across all countries precipitating an unprecedented demand for simultaneous curative, preventative, promotive, and curative services which has impacted the entire healthcare system. Even as it continues to rage across the globe, it is pertinent to learn from current experiences to build resilience into the system, especially for the vulnerable populations in rural areas.Methods: A retrospective cohort study of COVID-19 was conducted at COVID care centre in Vivekananda Memorial Hospital. A total of 136 COVID-19 confirmed patients were included in the study. Epidemiological and clinical profiles of patients were summarised. The association between risk factors and COVID-19 disease progression and patients’ outcomes at discharge were analysed and adjusted risk ratios were presented.Results: Out of the 136 patients, 128 were discharged uneventfully, 7 were referred to a higher centre because of disease progression and one patient died due to cardiac arrest.  The study showed statistically significant association between age above 50 years and the presence of diabetes with COVID-19 disease progression. Delayed presentation for care also carried a higher risk of negative outcomes. Significant number of diabetics had derangement of their glycaemic control during the period of study.Conclusions: The study has highlighted the importance of ensuring essential non-COVID public health services, especially targeted follow-up of older adults, people with diabetes for their treatment adherence and early detection and treatment initiation of COVID-19 amongst them.