SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Ubiquitin-mediated proteasomal degradation is a post-transcriptional protein modification that is comprised of various components including the 76-amino acid protein ubiquitin (Ub), Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), ubiquitin ligase (E3), deubiquitinating enzyme (DUB) and proteasome. We and others have recently provided genetic evidence showing that E3-ubiquitin ligases are associated with bone metabolism, the immune system and inflammation through ubiquitylation and subsequent degradation of their substrates. Dysregulation of the E3-ubiquitin ligase RNF146-mediated degradation of the adaptor protein 3BP2 (SH3 domain-binding protein 2) causes cherubism, an autosomal dominant disorder associated with severe inflammatory craniofacial dysmorphia syndrome in children. In this review, on the basis of our discoveries in cherubism, we summarize new insights into the roles of E3-ubiquitin ligases in the development of human disorders caused by an abnormal osteoimmune system by highlighting recent genetic evidence obtained in both human and animal model studies.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

Type 2 diabetes mellitus (T2DM) is a global health challenge. Therefore, understanding the molecular mechanisms underlying the pathophysiology of T2DM is key to improving current therapies. Loss of protein homeostasis leads to the accumulation of damaged proteins in cells, which results in tissue dysfunction. The elimination of damaged proteins occurs through the ubiquitin-proteasome system (UPS) and autophagy. In this review, we describe the mutual regulation between the UPS and autophagy and the involvement of these two proteolytic systems in metabolic dysregulation, insulin resistance, and T2DM. We propose that alterations in the UPS or autophagy contribute to triggering insulin resistance and the development of T2DM. In addition, these two pathways emerge as promising therapeutic targets for improving insulin resistance.

A mammalian soleus muscle along with other \"axial\" muscles ensures the stability of the body under the Earth\'s gravity. In rat experiments with hindlimb suspension, zero-gravity parabolic flights as well as in human dry immersion studies, a dramatic decrease in the electromyographic (EMG) activity of the soleus muscle has been repeatedly shown. Most of the motor units of the soleus muscle convert from a state of activity to a state of rest which is longer than under natural conditions. And the state of rest gradually converts to the state of disuse. This review addresses a number of metabolic events that characterize the earliest stage of the cessation of the soleus muscle contractile activity. One to three days of mechanical unloading are accompanied by energy-dependent dephosphorylation of AMPK, accumulation of the reactive oxygen species, as well as accumulation of resting myoplasmic calcium. In this transition period, a rapid rearrangement of the various signaling pathways occurs, which, primarily, results in a decrease in the rate of protein synthesis (primarily via inhibition of ribosomal biogenesis and activation of endogenous inhibitors of mRNA translation, such as GSK3β) and an increase in proteolysis (via upregulation of muscle-specific E3-ubiquitin ligases).

Since the discovery of ubiquitin conjugation as a cellular mechanism that triggers proteasomal degradation, the mode of substrate recognition by the ubiquitin-ligation system has been the holy grail of research in the field. This entails the discovery of recognition determinants within protein substrates, which are part of a degron, and explicit E3 ubiquitin (Ub)-protein ligases that trigger their degradation. Indeed, many protein substrates and their cognate E3\'s have been discovered in the past 40 years. In the course of these studies, various degrons have been randomly identified, most of which are acquired through post-translational modification, typically, but not exclusively, protein phosphorylation. Nevertheless, acquired degrons cannot account for the vast diversity in cellular protein half-life times. Obviously, regulation of the proteome is largely determined by inherent degrons, that is, determinants integral to the protein structure. Inherent degrons are difficult to predict since they consist of diverse sequence and secondary structure features. Therefore, unbiased methods have been employed for their discovery. This review describes the history of degron discovery methods, including the development of high throughput screening methods, state of the art data acquisition and data analysis. Additionally, it summarizes major discoveries that led to the identification of cognate E3 ligases and hitherto unrecognized complexities of degron function. Finally, we discuss future perspectives and what still needs to be accomplished towards achieving the goal of understanding how the eukaryotic proteome is regulated via coordinated action of components of the ubiquitin-proteasome system.

The Polycomb group (PcG) of proteins control developmental gene silencing and are highly conserved between flies and mammals. PcG proteins function by controlling post-translational modification of histones, such as ubiquitylation, which impacts chromatin compaction and thus gene transcription. Changes in PcG cellular levels have drastic effects on organismal development and are involved in the generation of human pathologies such as cancer. However, the mechanisms controlling their levels of expression and their physiological effects are only partially understood. In this work we describe the effects of modulating levels of SCE/dRING, a conserved E3 ubiquitin ligase and member of the PcG known to mono-ubiquitylate histone H2A. We find that inactivation of Sce induces apoptosis, an effect that is decreased in the absence of Dp53 function. However, over-expression of SCE produce no developmental effects but inhibits DP53-induced apoptosis. Thus, Sce functions as a Dp53-dependent apoptosis inhibitor. The SCE inhibition of DP53-induced apoptosis requires dRYBP, an ubiquitin binding protein and member of the PcG. Moreover, this inhibition of apoptosis involves the reduction of DP53 protein levels. Finally, high levels of SCE inhibit X-ray induced apoptosis as well as the apoptosis associated with tumor growth. We propose that SCE, together with dRYBP, inhibits apoptosis either by epigenetically regulating Dp53 transcription or by controlling the stabilization of DP53 protein levels thus promoting its ubiquitylation for proteaosomal degradation. This function may generate a homeostatic balance between apoptosis and proliferation during development that provides cell survival during the initiation and progression of disease processes.

To clarify the molecular changes of sublesional muscle in the acute phase of spinal cord injury (SCI), a moderately severe injury (40 g cm) was induced in the spinal cord (T10 vertebral level) of adult male Sprague-Dawley rats (injury) and compared with sham (laminectomy only). Rats were sacrificed at 48 h (acute) post injury, and gastrocnemius muscles were excised. Morphological examination revealed no significant changes in the muscle fiber diameter between the sham and injury rats. Western blot analyses performed on the visibly red, central portion of the gastrocnemius muscle showed significantly higher expression of muscle specific E3 ubiquitin ligases (muscle ring finger-1 and muscle atrophy f-box) and significantly lower expression of phosphorylated Akt-1/2/3 in the injury group compared to the sham group. Cyclooxygenase 2, tumor necrosis factor alpha (TNF-α), and caspase-1, also had a significantly higher expression in the injury group; although, the mRNA levels of TNF-α and IL-6 did not show any significant difference between the sham and injury groups. These results suggest activation of protein degradation, deactivation of protein synthesis, and development of inflammatory reaction occurring in the sublesional muscles in the acute phase of SCI before overt muscle atrophy is seen.