ABSTRACT:Oncogenic growth places great strain and dependence on protein homeostasis (proteostasis). This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for the frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for the treatment of other cancers at tolerable doses, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to their inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and increased autophagic flux to preserve proteostasis. Genetic inactivation of HSF1 sensitized AML cells to proteasome inhibition, marked by accumulation of unfolded protein, activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo. Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed human AML cells, and significantly reduced AML burden and extended survival in vivo. Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and colony formation and induced apoptosis in cells from patients with primary AML, including AML stem/progenitor cells, compared with normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition activated a terminal integrated stress response, which was surprisingly PKR. These studies unravel how proteostasis pathways are coopted to promote AML growth, progression and drug resistance and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

Tumor-infiltrating lymphocytes (TIL) often fail to restrain tumor growth due to progressive differentiation into an "exhausted" state. Tissue-resident memory T cells (TRM) maintain protection from infection for years in healthy tissues, and patient tumors that contain TIL with TRM features are associated with better prognosis. Proteomic and transcriptomic profiling of T cell populations identified proteostasis as a significant factor distinguishing TRM and progenitor-exhausted TIL from terminally exhausted TIL, including loss of E3 ubiquitin ligases NEURL3, RNF149, and WSB1, with accumulation of unfolded proteins despite functional proteasome activity. Enforced expression of these ligases in T cells preserved stem-like TCF1+ populations and improved function in tumors and chronic infection, whereas deficiency impaired TIL and altered T cell differentiation during acute infection. Sustained ligase expression rescued the accumulation of unfolded proteins in TIL and improved immunotherapy outcomes in preclinical models, underscoring the critical role of proteostasis in TIL function and highlighting a promising avenue for advancing cancer immunotherapy.

Translation elongation stalls trigger mRNA decay and degradation of the nascent polypeptide via translation-dependent quality control pathways. One such pathway, non-stop mRNA decay (NSD), targets aberrant mRNAs that lack stop codons, for example, due to premature polyadenylation. Here we identify Angel1, a CCR4 deadenylase homolog whose biochemical activity remains poorly defined, as a rate-limiting factor for NSD in human cells. Angel1 associates with mRNA coding regions and proteins involved in ribosome-associated quality control and mRNA decay, consistent with a factor that monitors translation elongation stalls. Depletion of Angel1 causes stabilization of reporter mRNAs that are targeted for NSD by the absence of stop codons, but not an mRNA targeted for nonsense-mediated decay. A conserved catalytic residue of Angel1 is critical for its function in NSD. Our findings identify Angel1 as a human NSD factor and suggest that Angel1 catalytic activity plays a critical role in the NSD pathway.

Proximity labeling approaches have been widely utilized to define protein interactomes. Due to the inherent promiscuity of proximity labeling using TurboID-based approaches, identification and adoption of appropriate labeling controls is a pivotal step to mitigate background interference and enhance interactome assignment accuracy. Here, we evaluate the effectiveness of both expression controls and data normalization strategies in generating high-confidence interactome maps. We demonstrate that the extent of control of TurboID protein expression is strongly correlated with overall signal intensity and the number of identified proteins from streptavidin-enrichments. Discordant expression levels between the bait and control samples result in high-frequency false-negative and false-positive identifications. Data normalization strategies help correct these expression differences but also introduce data distortion for proteins with high or low endogenous expression. Using the ubiquitin ligases RNF10 and HUWE1 as bait proteins, we demonstrate that matching TurboID expression between control and bait proteins allows for similar sampling of non-specific interactions. Using a matched expression strategy results in significantly reduced background interference and increases the accuracy of interactome assignments. These results document the need to alter proximity-labeling experimental workflows to include the generation of matched expression controls to enhance proximity labeling proteomics interactome mapping robustness and reproducibility.

The initiation-specific ribosome-associated quality control pathway (iRQC) is activated when translation initiation complexes fail to transition to elongation-competent 80S ribosomes. Upon iRQC activation, RNF10 ubiquitylates the 40S proteins uS3 and uS5, which leads to 40S decay. How iRQC is activated in the absence of pharmacological translation inhibitors and what mechanisms govern iRQC capacity and activity remain unanswered questions. Here, we demonstrate that altering 60S:40S stoichiometry by disrupting 60S biogenesis triggers iRQC activation and 40S decay. Depleting the critical scanning helicase eIF4A1 impairs 40S ubiquitylation and degradation, indicating mRNA engagement is required for iRQC. We show that amino acid starvation conditions also stimulate iRQC-dependent 40S decay. We identify RIOK3 as a crucial iRQC factor that interacts with ubiquitylated 40S subunits to mediate degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC pathway activation, establishing a feedforward mechanism that regulates iRQC capacity and subsequent 40S decay.

Background:Hypertension is a major risk factor for chronic kidney disease (CKD) and disproportionately affects African American women, contributing to disparities in kidney health outcomes. The biological mechanisms connecting hypertension to reduced kidney function, particularly in understudied populations, remain poorly understood. This study leverages transcriptomic analyses to uncover shared molecular signatures associated with hypertension and kidney function, focusing on female-specific profiles. Methods:The study analyzed whole-blood mRNA sequencing data from a cohort of 344 African American women, divided equally into discovery (n = 172) and validation (n = 172) datasets, along with 147 African American men. Differential expression (DE) analyses were performed to identify mRNAs associated with hypertension and kidney function (measured as eGFR). Female-specific findings were determined by comparing results between females and males. Pathway enrichment analyses were subsequently conducted to link the identified mRNAs to key biological mechanisms. Results:Comparative analyses revealed unique transcriptomic profiles in females, underscoring the role of sex-specific factors in disease progression. DE analyses identified 95 female-specific genes associated with both hypertension and eGFR. Subsequent pathway enrichment analysis with the 95 genes revealed key pathways related to fibrosis, inflammation, lipid metabolism, and endothelial dysfunction. The list of 95 includes TGF-β1 and PNPLA2 implicated in fibrotic and metabolic dysregulation, and immune system players such as IL32 and TNFSF12 that amplify inflammation and kidney injury. Conclusions:This study provides novel insights into the transcriptomic mechanisms underlying hypertension and kidney function in African American women. The findings emphasize the importance of addressing sex-specific and population-specific molecular mechanisms to inform precision medicine approaches and reduce health disparities hypertension-related impaired kidney function. Future research should prioritize experimental validation and longitudinal studies to further elucidate these pathways.

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. To date, hundreds of thousands of ubiquitination sites (ubi-sites) have been identified, however fewer than 1% have known functional roles. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. To pinpoint critical regulatory events requiring ubiquitination at a precise site, we mapped ubi-site conservation across proteomics data from six non-human species. Perturbation proteomics revealed that highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize site-specific ubiquitination relevant for organismal fitness, we constructed a machine learning-based positional importance score for more than 100,000 ubi-sites, which identifies sites regulating diverse protein functions and rationalizes genetic vulnerabilities. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying site-specific protein ubiquitination.

Mitochondrial uncoupling in brown and beige adipocytes underlies non-shivering thermogenesis, during which cells exhibit elevated mitochondrial activity and protein synthesis. Here, we identify the ribosome-associated quality control (RQC) pathway, initiated by the ubiquitin ligase ZNF598, as essential for resolving translation conflicts and ribosome collisions that arise during this process. We demonstrate that β-adrenergic stimulation, cold exposure, and dietary changes induce RQC in adipocytes. ZNF598 is required for stimulated mitochondrial biogenesis, respiration, and uncoupling, and mice with adipocyte-specific Znf598 deletion display impaired adipose browning and metabolic dysfunction when obese. Conversely, induced overexpression of ZNF598 in adipocytes of adult mice enhances RQC capacity and improves adaptive adipose responses. These findings establish translational rewiring and ribosome collision as features of thermogenesis and RQC as a critical mediation strategy. ### Competing Interest Statement The authors have declared no competing interest.

Aging is one of the most prominent risk factors for neurodegeneration, yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging, we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons, which retained their aging hallmarks, and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins, especially spliceosome components. Intriguingly, splicing proteins-like the dementia- and ALS-associated protein TDP-43-mislocalize to the cytoplasm in aged neurons, which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules, but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery, poor HSP90α chaperone activity and the failure to respond to new stress events. Together, our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons.

Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation. We demonstrate that increased accumulation of cytoplasmic double-stranded RNA (dsRNA) activates the eIF2α kinase PKR, which in turn triggers the ISR in aged neurons and leads to sequestration of dsRNA in stress granules. Aged neurons accumulate endogenous mitochondria-derived dsRNA that directly binds to PKR. This mitochondrial dsRNA leaks through damaged mitochondrial membranes and forms cytoplasmic foci in aged neurons. Finally, we demonstrate that PKR inhibition leads to the cessation of stress, resumption of cellular translation, and restoration of RNA-binding protein expression. Together, our results identify a source of RNA stress that destabilizes aged neurons and may contribute to neurodegeneration.

Potassium channels (Kv) are responsible for repolarizing the action potential in cardiomyocytes. There is a variety of Kv isoforms and corresponding currents (e.g. IKto, IKslow1, IKslow2) that contribute to different phases of repolarization. Because only the sum of their activities can be measured in the form of currents (IKsum), there is a need to delineate individual K+ currents. Most existing studies make inference of Kv activities via curve-fitting procedures but encounter certain limitations as follows: (1) curve-fitting decomposition only relies on the shape of K+ current traces, which does not discern the underlying kinetics; (2) IKsum traces can only be fitted for one clamp voltage at each time, and then analyzed in a population-averaged way later. This paper presents a novel concurrent data assimilation method to calibrate biophysics-based models and delineate kinetics of Kv isoforms with multiple voltage-clamp responses simultaneously. The proposed method is evaluated and validated with whole-cell IKsum recordings from wild-type and chronically glycosylation-deficient cardiomyocytes. Experimental results show that the proposed method effectively handles multiple-response data and describes glycosylation-conferred perturbations to Kv isoforms. Further, we develop a graphical-user-interface (GUI) application that provides an enabling tool to biomedical scientists for data-driven modeling and analysis of Kv kinetics in various heart diseases.

Hematopoietic stem cells (HSCs) give rise to all blood and immune cells throughout life. Aging HSCs exhibit diminished regenerative function, myeloid-biased differentiation, and clonal outgrowth, which contribute to compromised immunity and increased incidence of bone marrow failure and hematological malignancies in older adults. Interventions that prevent or mitigate age-related stem cell exhaustion could thus have an outsized impact on extending human healthspan and longevity. Loss of proteostasis is a hallmark of aging and can impair HSC self-renewal, but whether interventions that enhance proteostasis capacity improve HSC fitness and mitigate age-related changes in HSC function is untested. To test this hypothesis, we generated a mouse model with high-fidelity ribosomes (RiboHiFi) that reduces translation errors during protein synthesis. Strikingly, elevated translation fidelity protects HSCs from the detrimental effects of increased protein synthesis activity and extends HSC longevity in vivo. HSCs from old (24-30 month) adult RiboHiFi mice exhibit reduced proteostasis dysfunction, as evidenced by lower expression of several proteostasis stress response factors. Furthermore, elevated translation fidelity delays stem cell exhaustion in serial transplantation experiments, with some old adult RiboHiFi HSCs retaining long-term multilineage reconstituting activity through six rounds of transplantation. Overall, these studies reveal an opportunity for enhancing translation fidelity to promote stem cell fitness, stress resistance, and longevity.

Translation control mechanisms connect the largely static genome to the highly dynamic proteome. At each step in the translation cycle, multiple layers of regulation enable efficient protein biogenesis under optimal conditions and mediate responses to acute environmental challenges. Recent research has demonstrated that individual ribosomal protein ubiquitylation events act as molecular signals to specify quality control pathway outcomes. Here, we synthesize current knowledge of ubiquitin-mediated translation control mechanisms and highlight key outstanding questions. We compare and contrast ubiquitin-dependent mechanisms that regulate ribosome-associated quality control pathways at several steps in the translation cycle. We also explore how distinct ribosome ubiquitylation events on specific ribosomal proteins impact translation activity and how defects in specific ubiquitin-mediated regulatory steps impact physiology and health.

Background Heart failure (HF) presents a massive burden to health care with a complex pathophysiology that results in HF with reduced left ventricle ejection fraction (EF) or HF with preserved EF. It has been shown that relatively modest changes in protein glycosylation, an essential posttranslational modification, are associated with clinical presentations of HF. We and others previously showed that such aberrant protein glycosylation in animal models can lead to HF. Methods and Results We develop and characterize a novel, tamoxifen‐inducible, cardiomyocyte Mgat1 knockout mouse strain, achieved through deletion of Mgat1 , alpha‐1,3‐mannosyl‐glycoproten 2‐beta‐N‐acetlyglucosaminyltransferase, which encodes N‐acetylglucosaminyltransferase I. We investigate the role of hybrid/complex N‐glycosylation in adult HFrEF pathogenesis at the ion channel, cardiomyocyte, tissue, and gross cardiac level. The data demonstrate successful reduction of N‐acetylglucosaminyltransferase I activity and confirm that hybrid/complex N‐glycans modulate gating of cardiomyocyte voltage‐gated calcium channels. A longitudinal study shows that the tamoxifen‐inducible, cardiomyocyte Mgat1 knockout mice present with significantly reduced systolic function by 28 days post induction that progresses into HFrEF by 8 weeks post induction, without significant ventricular dilation or hypertrophy. Further, there was minimal, if any, physiologic or pathophysiologic cardiomyocyte electromechanical remodeling or fibrosis observed before (10–21 days post induction) or after (90–130 days post induction) HFrEF development. Conclusions The tamoxifen‐inducible, cardiomyocyte Mgat1 knockout mouse strain created and characterized here provides a model to describe novel mechanisms and causes responsible for HFrEF onset in the adult, likely occurring primarily through tissue‐level reductions in electromechanical activity in the absence of (or at least before) cardiomyocyte remodeling and fibrosis.

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

In vitro cytotoxicity of PF-06804103 across a panel of cancer cell lines.

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIβ isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

ADC retention times by hydrophobic interaction chromatography (HIC).

In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model.