Systematic Analysis of Ribophagy in Human Cells Reveals By-stander Flux During Selective Autophagy

Ribosomes are abundant cellular machines1,2 regulated by assembly, supernumerary subunit turnover, and nascent chain quality control mechanisms1–5. Moreover, nitrogen starvation in yeast has been reported to promote selective ribosome delivery to the vacuole in an autophagy conjugation system-dependent manner, a process called “ribophagy”6,7. However, whether ribophagy in mammals is selective or regulated is unclear. Using Ribo-Keima flux reporters, we find that starvation or mTOR inhibition promotes VPS34-dependent ribophagic flux, which unlike yeast, is largely ATG8 conjugation independent and occurs concomitantly with other cytosolic protein autophagic flux reporters8,9. Ribophagic flux was not induced upon inhibition of translational elongation or nascent chain uncoupling, but was induced in a comparatively selective manner upon proteotoxic stress via arsenite10 or chromosome mis-segregation11 dependent upon VPS34 and ATG8 conjugation. Unexpectedly, agents typically used to induce selective autophagy also promoted increased ribosome and cytosolic protein reporter flux, suggesting significant bulk or “by-stander” autophagy during what is often considered selective autophagy12,13. These results emphasize the importance of monitoring non-specific cargo flux when assessing selective autophagy pathways

Development of Activity-Based Probes for Ubiquitin and Ubiquitin-like Protein Signaling Pathways

Ubiquitin and ubiquitin-like (UBL) proteins regulate a vast variety of cellular functions. Some UBL proteins are present in all cell types, while others are expressed only in certain cells or under certain environmental conditions. This highlights the central role of UBL systems in regulation of ubiquitous as well as specific cellular functions. UBL proteins share little amino acid sequence identity to each other, yet they share similar 3D shapes, which is exemplified by the β-grasp fold. Central to UBL protein signaling pathways are UBL protein-activating E1 enzymes that activate the C-terminus of UBL proteins for subsequent conjugation to the protein substrates. Due to their critical roles in biology, E1 enzymes have been recognized as emerging drug targets to treat human diseases. In spite of their biological significance, however, methods to discover UBL proteins and to monitor the intracellular activity of E1 enzymes are lacking. Thus, there is a critical need for methods to evaluate the intracellular mechanisms of action of E1 enzyme inhibitors. Here we describe the development of a mechanism-based small-molecule probe, <b>ABP1</b>, that can be used to discover and to detect active UBL proteins, and to monitor the intracellular activity of E1 enzymes inside intact cells. The developed probe can also be used to profile the selectivity of E1 enzyme-targeting drugs <i>in vitro</i> and inside intact cells

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development. Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.</p