Proteomic analysis of lung responses to SARS-CoV-2 infection in aged non-human primates: clinical and research relevance.

With devastating health and socioeconomic impact worldwide, much work is left to understand the Coronavirus Disease 2019 (COVID-19), with emphasis in the severely affected elderly population. Here, we present a proteomics study of lung tissue obtained from aged vs. young rhesus macaques (Macaca mulatta) and olive baboons (Papio Anubis) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using age as a variable, we identified common proteomic profiles in the lungs of aged infected non-human primates (NHPs), including key regulators of immune function, as well as cell and tissue remodeling, and discuss the potential clinical relevance of such parameters. Further, we identified key differences in proteomic profiles between both NHP species, and compared those to what is known about SARS-CoV-2 in humans. Finally, we explored the translatability of these animal models in the context of aging and the human presentation of the COVID-19.

Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Integrative multi-omics characterization of hepatocellular carcinoma in Hispanic patients.

Background: The incidence and mortality rates of hepatocellular carcinoma (HCC) among Hispanics in the United States are much higher than those of non-Hispanic whites. We conducted comprehensive multi-omics analyses to understand molecular alterations in HCC among Hispanic patients. Methods: Paired tumor and adjacent non-tumor samples were collected from 31 Hispanic HCC in South Texas (STX-Hispanic) for genomic, transcriptomic, proteomic, and metabolomic profiling. Additionally, serum lipids were profiled in 40 Hispanic and non-Hispanic patients with or without clinically diagnosed HCC. Results: Exome sequencing revealed high mutation frequencies of AXIN2 and CTNNB1 in STX Hispanic HCCs, suggesting a predominant activation of the Wnt/ß-catenin pathway. The TERT promoter mutation frequency was also remarkably high in the Hispanic cohort. Cell cycles and liver functions were identified as positively- and negatively-enriched, respectively, with gene set enrichment analysis. Gene sets representing specific liver metabolic pathways were associated with dysregulation of corresponding metabolites. Negative enrichment of liver adipogenesis and lipid metabolism corroborated with a significant reduction in most lipids in the serum samples of HCC patients. Two HCC subtypes from our Hispanic cohort were identified and validated with the TCGA liver cancer cohort. The subtype with better overall survival showed higher activity of immune and angiogenesis signatures, and lower activity of liver function-related gene signatures. It also had higher levels of immune checkpoint and immune exhaustion markers. Conclusions: Our study revealed some specific molecular features of Hispanic HCC and potential biomarkers for therapeutic management of HCC and provides a unique resource for studying Hispanic HCC.

SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis.

RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. SERBP1 is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. Using a proteomics approach followed by functional analysis, we defined SERBP1's interactome. We uncovered novel SERBP1 roles in splicing, cell division, and ribosomal biogenesis and showed its participation in pathological stress granules and Tau aggregates in Alzheimer's disease brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.

HIV infection impairs the host response to Mycobacterium tuberculosis infection by altering surfactant protein D function in the human lung alveolar mucosa.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

The 2023 Report on the Proteome from the HUPO Human Proteome Project.

Since 2010, the Human Proteome Project (HPP), the flagship initiative of the Human Proteome Organization (HUPO), has pursued two goals: (1) to credibly identify the protein parts list and (2) to make proteomics an integral part of multiomics studies of human health and disease. The HPP relies on international collaboration, data sharing, standardized reanalysis of MS data sets by PeptideAtlas and MassIVE-KB using HPP Guidelines for quality assurance, integration and curation of MS and non-MS protein data by neXtProt, plus extensive use of antibody profiling carried out by the Human Protein Atlas. According to the neXtProt release 2023-04-18, protein expression has now been credibly detected (PE1) for 18,397 of the 19,778 neXtProt predicted proteins coded in the human genome (93%). Of these PE1 proteins, 17,453 were detected with mass spectrometry (MS) in accordance with HPP Guidelines and 944 by a variety of non-MS methods. The number of neXtProt PE2, PE3, and PE4 missing proteins now stands at 1381. Achieving the unambiguous identification of 93% of predicted proteins encoded from across all chromosomes represents remarkable experimental progress on the Human Proteome parts list. Meanwhile, there are several categories of predicted proteins that have proved resistant to detection regardless of protein-based methods used. Additionally there are some PE1-4 proteins that probably should be reclassified to PE5, specifically 21 LINC entries and ∼30 HERV entries; these are being addressed in the present year. Applying proteomics in a wide array of biological and clinical studies ensures integration with other omics platforms as reported by the Biology and Disease-driven HPP teams and the antibody and pathology resource pillars. Current progress has positioned the HPP to transition to its Grand Challenge Project focused on determining the primary function(s) of every protein itself and in networks and pathways within the context of human health and disease.

Multi-omics analyses reveal novel effects of PLCγ2 deficiency in the mouse brain.

Phospholipase C gamma-2 (PLCγ2) catalyzes the hydrolysis of the membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into numerous downstream signaling pathways. PLCG2 polymorphisms are associated with both reduced and increased risk of Alzheimer's disease (AD) and with longevity. In the brain, PLCG2 is highly expressed in microglia, where it is proposed to regulate phagocytosis, secretion of cytokines/chemokines, cell survival and proliferation. We analyzed the brains of three-month-old PLCγ2 knockout (KO), heterozygous (HET), and wild-type (WT) mice using multiomics approaches, including shotgun lipidomics, proteomics, and gene expression profiling, and immunofluorescence. Lipidomic analyses revealed sex-specific losses of total cerebrum PIP2 and decreasing trends of DAG content in KOs. In addition, PLCγ2 depletion led to significant losses of myelin-specific lipids and decreasing trends of myelin-enriched lipids. Consistent with our lipidomics results, RNA profiling revealed sex-specific changes in the expression levels of several myelin-related genes. Further, consistent with the available literature, gene expression profiling revealed subtle changes on microglia phenotype in mature adult KOs under baseline conditions, suggestive of reduced microglia reactivity. Immunohistochemistry confirmed subtle differences in density of microglia and oligodendrocytes in KOs. Exploratory proteomic pathway analyses revealed changes in KO and HET females compared to WTs, with over-abundant proteins pointing to mTOR signaling, and under-abundant proteins to oligodendrocytes. Overall, our data indicate that loss of PLCγ2 has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology and aging via novel mechanisms in addition to regulation of microglia function.

The Association of Biomolecular Resource Facilities Proteome Informatics Research Group Study on Metaproteomics (iPRG-2020).

Metaproteomics research using mass spectrometry data has emerged as a powerful strategy to understand the mechanisms underlying microbiome dynamics and the interaction of microbiomes with their immediate environment. Recent advances in sample preparation, data acquisition, and bioinformatics workflows have greatly contributed to progress in this field. In 2020, the Association of Biomolecular Research Facilities Proteome Informatics Research Group launched a collaborative study to assess the bioinformatics options available for metaproteomics research. The study was conducted in 2 phases. In the first phase, participants were provided with mass spectrometry data files and were asked to identify the taxonomic composition and relative taxa abundances in the samples without supplying any protein sequence databases. The most challenging question asked of the participants was to postulate the nature of any biological phenomena that may have taken place in the samples, such as interactions among taxonomic species. In the second phase, participants were provided a protein sequence database composed of the species present in the sample and were asked to answer the same set of questions as for phase 1. In this report, we summarize the data processing methods and tools used by participants, including database searching and software tools used for taxonomic and functional analysis. This study provides insights into the status of metaproteomics bioinformatics in participating laboratories and core facilities.

Pharmacological inhibition of KDM1A/LSD1 enhances estrogen receptor beta-mediated tumor suppression in ovarian cancer.

Ovarian cancer (OCa) is the most lethal gynecologic cancer. Emerging data indicates that estrogen receptor beta (ERß) functions as a tumor suppressor in OCa. Lysine-specific histone demethylase 1A (KDM1A) is an epigenetic modifier that acts as a coregulator for steroid hormone receptors. However, it remain unknown if KDM1A interacts with ERß and regulates its expression/functions in OCa. Analysis of TCGA data sets indicated KDM1A and ERß expression showed an inverse relationship in OCa. Knockout (KO), knockdown (KD), or inhibition of KDM1A increased ERß isoform 1 expression in established and patient-derived OCa cells. Further, KDM1A interacts with and functions as a corepressor of ERß, and its inhibition enhances ERß target gene expression via alterations of histone methylation marks at their promoters. Importantly, KDM1A-KO or -KD enhanced the efficacy of ERß agonist LY500307, and the combination of KDM1A inhibitor (KDM1Ai) NCD38 with ERß agonist synergistically reduced the cell viability, colony formation, and invasion of OCa cells. RNA-seq and DIA mass spectrometry analyses showed that KDM1A-KO resulted in enhanced ERß signaling and that genes altered by KDM1A-KO and ERß agonist were related to apoptosis, cell cycle, and EMT. Moreover, combination treatment significantly reduced the tumor growth in OCa orthotopic, syngeneic, and patient-derived xenograft models and proliferation in patient-derived explant models. Our results demonstrate that KDM1A regulates ERß expression/functions, and its inhibition improves ERß mediated tumor suppression. Overall, our findings suggest that KDM1Ai and ERß agonist combination therapy is a promising strategy for OCa.

Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain.

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.

Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Siphophage 0105phi7-2 of Bacillus thuringiensis: Novel Propagation, DNA, and Genome-Implied Assembly.

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head-host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.

Mass Spectral Analyses of Salmonella Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids.

Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma.

Retinoblastoma is a cancer of the infant retina primarily driven by loss of the Rb tumor suppressor gene, which is undruggable. Here, we report an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell-surface receptor for SFRP2 in retinoblastoma cells; that CXADR functions as a "dependence receptor," transmitting a growth-inhibitory signal in the absence of SFRP2; and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induces nitric oxide and profoundly inhibits retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation and is amenable to therapeutic intervention.

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project.

The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 407 (93.2%) of the 19 750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from data sets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the human proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Structural and functional asymmetry of RING trimerization controls priming and extension events in TRIM5α autoubiquitylation.

TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.

Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure.

Accumulating evidence indicates that extracellular vesicles (EVs) mediate endocrine functions and also pathogenic effects of neurodevelopmental perturbagens like ethanol. We performed mass-spectrometry on EVs secreted by fetal murine cerebral cortical neural stem cells (NSCs), cultured ex-vivo as sex-specific neurosphere cultures, to identify overrepresented proteins and signaling pathways in EVs relative to parental NSCs in controls, and following exposure of parental NSCs to a dose range of ethanol. EV proteomes differ substantially from parental NSCs, and though EVs sequester proteins across sub-cellular compartments, they are enriched for distinct morphogenetic signals including the planar cell polarity pathway. Ethanol exposure favored selective protein sequestration in EVs and depletion in parental NSCs, and also resulted in dose-independent overrepresentation of cell-cycle and DNA replication pathways in EVs as well as dose-dependent overrepresentation of rRNA processing and mTor stress pathways. Transfer of untreated EVs to naïve cells resulted in decreased oxidative metabolism and S-phase, while EVs derived from ethanol-treated NSCs exhibited diminished effect. Collectively, these data show that NSCs secrete EVs with a distinct proteome that may have a general growth-inhibitory effect on recipient cells. Moreover, while ethanol results in selective transfer of proteins from NSCs to EVs, the efficacy of these exposure-derived EVs is diminished.

Targeting LIPA independent of its lipase activity is a therapeutic strategy in solid tumors via induction of endoplasmic reticulum stress.

Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.