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Aims: The knowledge of the molecular players that regulate the generation of endoderm cells is imperative to obtain homogenous population of pancreatic β-cells from stem cells. The Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, but its role in the generation of β-cells remains elusive. Hence, it would be appropriate to unravel the potential role of UPS in endoderm specification and utilize the understanding to generate β-cells from pluripotent stem cells., Materials and Methods: The pluripotent stem cells (mESCs, miPSCs and hIPSCs) were subjected to differentiation towards pancreatic β-cells and assessed the proteasomal activity during endodermal differentiation. Pharmacologic agents MG132 and IU-1 were employed to inhibit and activate proteasomal activity respectively at the definitive endoderm stage to investigate its impact on the generation of β-cells. The expression of stage-specific genes were analyzed at transcript and protein levels. We also explored the role of unfolded protein response and UPS-regulated signalling pathways in endodermal differentiation., Key Findings: We observed decreased proteasomal activity specifically during endoderm, but not during the generation of other lineages. Extraneous proteasomal inhibition enhanced the expression of endodermal genes while increasing the proteasomal activity hindered definitive endodermal differentiation. Proteasomal inhibition at the definitive endodermal stage culminated in an enriched generation of insulin-positive cells. Elevated endodermal gene expression was consistent in mESCs and hIPSCs upon proteasomal inhibition. Mechanistic insight revealed the proteasome-inhibited enhanced endodermal differentiation to be via modulating the YAP pathway., Significance: Our study unravels the specific involvement of UPS in endoderm cell generation from pluripotent stem cells and paves the way for obtaining potential definitive endodermal cells for plausible cellular therapy in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ubiquitination defects have been reported in various diseases, including neurodegenerative diseases, metabolic disorders and cancer. Balance between degradation and synthesis of the proteins to treat cancer can be managed by designing a chimeric molecule, known as Proteolysis Targeting Chimeric molecule (Lee, Kim et al. 2021). Proteolysis-targeting chimeras (PROTACs) acts as a tool for conducting therapeutic intervention. It eradicates or reduces the proteins that are responsible for causing diseases. Each PROTAC contains a target warhead, an E3 ligand and a linker. E3 ligases are recruited by these bifunctional molecules, and the Ubiquitin (Ub) Proteasome System (UPS) is used to target the degradation of specific proteins. As compared to inhibition, this degradation offers several advantages in the drug resistance, selectivity, and potency. Thus, numerous small molecule PROTACs are identified so far. In this review, the development of PROTACs, historical milestones, the biological mechanism, advantages and recent progress, and role of PROTAC in prostate cancer, breast cancer, non-hodgkin lymphoma, multiple myeloma, and malignant peripheral nerve sheath tumors are summarized., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative disease characterized by motor and non-motor symptoms. Impairment of the ubiquitin proteasome system (UPS) and autophagy has been suggested to contribute to α-synuclein accumulation, which is identified as the pathological hallmark of PD. Recently, alteration in histone-3 acetylation has also been found to be correlated to PD. Interestingly, the histone deacetylase 6 (HDAC6) enzyme, which regulates the acetylation of histone-3, was shown to be involved in autophagy. Venlafaxine is an antidepressant that was proposed to inhibit HDAC expression in depressive rats' hippocampi. In this study, we aimed to examine the ability of venlafaxine to inhibit striatal HDAC6 and to enhance α-synuclein clearance through the activation of the UPS and autophagy, in addition to treating depression, which is the most debilitating non-motor symptom, in a rotenone model of PD. Venlafaxine administration was noted to decrease α-synuclein accumulation and preserve dopaminergic neurons along with restoration of striatal dopamine levels and motor recovery. Its administration augmented the UPS and autophagic markers (beclin-1, p62, and LC3) with consequent modulation of apoptotic indicators (Bax/Bcl-2 ratio, cytochrome c, and caspase-3). Additionally, venlafaxine inhibited HDAC6 with further enhancement of autophagy and restoration of histone-3 acetylation with subsequent increases in survival gene expressions (Bcl-2 and brain-derived neurotrophic factor). Chloroquine (autophagy inhibitor) was used to indicate the proposed pathway. Moreover, venlafaxine hampered depressive symptoms and improved hippocampal noradrenaline and serotonin levels. Collectively, venlafaxine is suggested to display neuroprotective effects with improvement of motor and non-motor PD symptoms., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein., (Copyright © 2016 Elsevier Inc. All rights reserved.)