How degrading! Trapped translation factors get trashed.

Using small molecules that trap translation factors within translating ribosomes, Gurzeler et al.1 and Oltion et al.2 identify a new branch of the ribosome-associated quality-control (RQC) pathway. This mode of translation regulation expands the number of mechanistically distinct RQC pathways.

HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways.

HUWE1 is a large, enigmatic HECT-domain ubiquitin ligase implicated in the regulation of diverse pathways, including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we find that HUWE1 targets substrates in a largely cell-type-specific manner. However, we identify C16orf72/HAPSTR1 as a robust HUWE1 substrate in multiple cell lines. Previously established physical and genetic interactions between HUWE1 and HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is required for HUWE1 nuclear localization and nuclear substrate targeting. Nuclear HUWE1 is required for both cell proliferation and modulation of stress signaling pathways, including p53 and nuclear factor κB (NF-κB)-mediated signaling. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

The OPAQUE1/DISCORDIA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize.

Formative asymmetric divisions produce cells with different fates and are critical for development. We show the maize (Zea mays) myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells before and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 becomes apparent during late cytokinesis, when the phragmoplast forms the nascent cell plate. Initial phragmoplast guidance in o1 is normal; however, as phragmoplast expansion continues o1 phragmoplasts become misguided. To understand how O1 contributes to phragmoplast guidance, we identified O1-interacting proteins. Maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs), which are also required for correct phragmoplast guidance, physically interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

Hematopoietic stem cells preferentially traffic misfolded proteins to aggresomes and depend on aggrephagy to maintain protein homeostasis.

Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). However, how HSCs purge misfolded proteins is unknown. Here, we show that in contrast to most cells that primarily utilize the proteasome to degrade misfolded proteins, HSCs preferentially traffic misfolded proteins to aggresomes in a Bag3-dependent manner and depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. When autophagy is disabled, HSCs compensate by increasing proteasome activity, but proteostasis is ultimately disrupted as protein aggregates accumulate and HSC function is impaired. Bag3-deficiency blunts aggresome formation in HSCs, resulting in protein aggregate accumulation, myeloid-biased differentiation, and diminished self-renewal activity. Furthermore, HSC aging is associated with a severe loss of aggresomes and reduced autophagic flux. Protein degradation pathways are thus specifically configured in young adult HSCs to preserve proteostasis and fitness but become dysregulated during aging.

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Polarly localized WPR proteins interact with PAN receptors and the actin cytoskeleton during maize stomatal development.

Polarization of cells prior to asymmetric cell division is crucial for correct cell divisions, cell fate, and tissue patterning. In maize (Zea mays) stomatal development, the polarization of subsidiary mother cells (SMCs) prior to asymmetric division is controlled by the BRICK (BRK)-PANGLOSS (PAN)-RHO FAMILY GTPASE (ROP) pathway. Two catalytically inactive receptor-like kinases, PAN2 and PAN1, are required for correct division plane positioning. Proteins in the BRK-PAN-ROP pathway are polarized in SMCs, with the polarization of each protein dependent on the previous one. As most of the known proteins in this pathway do not physically interact, possible interactors that might participate in the pathway are yet to be described. We identified WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1)/PLASTID MOVEMENT IMPAIRED 2 (PMI2)-RELATED (WPR) proteins as players during SMC polarization in maize. WPRs physically interact with PAN receptors and polarly accumulate in SMCs. The polarized localization of WPR proteins depends on PAN2 but not PAN1. CRISPR-Cas9-induced mutations result in division plane defects in SMCs, and ectopic expression of WPR-RFP results in stomatal defects and alterations to the actin cytoskeleton. We show that certain WPR proteins directly interact with F-actin through their N-terminus. Our data implicate WPR proteins as potentially regulating actin filaments, providing insight into their molecular function. These results demonstrate that WPR proteins are important for cell polarization.

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

Persistent mRNA localization defects and cell death in ALS neurons caused by transient cellular stress.

Persistent cytoplasmic aggregates containing RNA binding proteins (RBPs) are central to the pathogenesis of late-onset neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). These aggregates share components, molecular mechanisms, and cellular protein quality control pathways with stress-induced RNA granules (SGs). Here, we assess the impact of stress on the global mRNA localization landscape of human pluripotent stem cell-derived motor neurons (PSC-MNs) using subcellular fractionation with RNA sequencing and proteomics. Transient stress disrupts subcellular RNA and protein distributions, alters the RNA binding profile of SG- and ALS-relevant RBPs and recapitulates disease-associated molecular changes such as aberrant splicing of STMN2. Although neurotypical PSC-MNs re-establish a normal subcellular localization landscape upon recovery from stress, cells harboring ALS-linked mutations are intransigent and display a delayed-onset increase in neuronal cell death. Our results highlight subcellular molecular distributions as predictive features and underscore the utility of cellular stress as a paradigm to study ALS-relevant mechanisms.

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.

Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition.

HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection.

Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome-associated quality control (RQC) factor ZNF598 during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 (RPS20) and eS10 (RPS10), initiating RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection enhances uS10 ubiquitylation, indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells that either lack ZNF598 or express a ubiquitylation-deficient version of uS10. Using SILAC-based proteomics and concurrent RNA-seq analysis, we determine that translation, but not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

EDF1 coordinates cellular responses to ribosome collisions.

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions.

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification of putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

PD-1 and BTLA regulate T cell signaling differentially and only partially through SHP1 and SHP2.

Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.

Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role and fate of ubiquitylated proteins remain unclear. We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation, respectively, suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes. We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation. Critically, cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway.

Ribosomes are cellular machines that build proteins by latching on and then reading template molecules known as mRNAs. Several ribosomes may be moving along the same piece of mRNA at the same time, each making their own copy of the same protein. Damage to an mRNA or other problems may cause a ribosome to stall, leading to subsequent collisions. A quality control pathway exists to identify stalled ribosomes and fix the 'traffic jams'. It relies on enzymes that tag halted ribosomes with molecules known as ubiquitin. The cell then removes these ribosomes from the mRNA and destroys the proteins they were making. Afterwards, additional enzymes take off the ubiquitin tags so the cell can recycle the ribosomes. These enzymes are key to signaling the end of the quality control event, yet their identity was still unclear. Garshott et al. used genetic approaches to study traffic jams of ribosomes in mammalian cells. The experiments showed that cells added sets of ubiquitin tags to stalled ribosomes in a specific order. Two enzymes, known as USP21 and OTUD3, could stop this process; this allowed ribosomes to carry on reading mRNA. Further work revealed that the ribosomes in cells that produce higher levels of USP21 and OTUD3 were less likely to stall on mRNA. On the other hand, ribosomes in cells lacking USP1 and OTUD3 retained their ubiquitin tags for longer and were more likely to stall. The findings of Garshott et al. reveal that USP21 and OTUD3 are involved in the quality control pathway which fixes ribosome traffic jams. In mice, problems in this pathway have been linked with neurons dying or being damaged because toxic protein products start to accumulate in cells; this is similar to what happens in human conditions such as Alzheimer's and Parkinson's diseases. Using ubiquitin to target and potentially fix the pathway could therefore open the door to new therapies.

Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics.

Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics. We show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Critically, inhibiting neither UAE nor NAE significantly affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Using antibodies with varying preference for free ubiquitin or polyubiquitin and fluorescently tagged ubiquitin variants in combination with UAE inhibition, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions.

Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response.

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance.

Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome.

Protein ubiquitylation is one of the most prevalent posttranslational modifications (PTM) within cells. Ubiquitin modification of target lysine residues typically marks substrates for proteasome-dependent degradation. However, ubiquitylation can also alter protein function through modulation of protein complexes, localization, or activity, without impacting protein turnover. Taken together, ubiquitylation imparts critical regulatory control over nearly every cellular, physiological, and pathophysiological process. Affinity purification techniques coupled with quantitative mass spectrometry have been robust tools to identify PTMs on endogenous proteins. A peptide antibody-based affinity approach has been successfully utilized to enrich for and identify endogenously ubiquitylated proteins. These antibodies recognize the Lys-ϵ-Gly-Gly (diGLY) remnant that is generated following trypsin digestion of ubiquitylated proteins, and these peptides can then be identified by standard mass spectrometry approaches. This technique has led to the identification of >50,000 ubiquitylation sites in human cells and quantitative information about how many of these sites are altered upon exposure to diverse proteotoxic stressors. In addition, the diGLY proteomics approach has led to the identification of specific ubiquitin ligase targets. Here we provide a detailed method to interrogate the ubiquitin-modified proteome from any eukaryotic organism or tissue.