ProEnd: A Comprehensive Database for Identifying HbYX Motif-Containing Proteins Across the Tree of Life.

The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were tested and found to directly interact with the proteasome. ProEnd's extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer. Link: http://proend.org/.

Proteasome hyperactivation rewires the proteome enhancing stress resistance, proteostasis, lipid metabolism and ERAD in C. elegans.

Proteasome dysfunction is implicated in the pathogenesis of neurodegenerative diseases and age-related proteinopathies. Using a C. elegans model, we demonstrate that 20S proteasome hyperactivation, facilitated by 20S gate-opening, accelerates the targeting of intrinsically disordered proteins. This leads to increased protein synthesis, extensive rewiring of the proteome and transcriptome, enhanced oxidative stress defense, accelerated lipid metabolism, and peroxisome proliferation. It also promotes ER-associated degradation (ERAD) of aggregation-prone proteins, such as alpha-1 antitrypsin (ATZ) and various lipoproteins. Notably, our results reveal that 20S proteasome hyperactivation suggests a novel role in ERAD with broad implications for proteostasis-related disorders, simultaneously affecting lipid homeostasis and peroxisome proliferation. Furthermore, the enhanced cellular capacity to mitigate proteostasis challenges, alongside unanticipated acceleration of lipid metabolism is expected to contribute to the longevity phenotype of this mutant. Remarkably, the mechanism of longevity induced by 20S gate opening appears unique, independent of known longevity and stress-resistance pathways. These results support the therapeutic potential of 20S proteasome activation in mitigating proteostasis-related disorders broadly and provide new insights into the complex interplay between proteasome activity, cellular health, and aging.

Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses.

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigated the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.