Affinity Purification Mass Spectrometry Research Articles

Nuclear Factor I family members are key transcription factors regulating gene expression

The Nuclear Factor I (NFI) family of transcription factors (TFs) plays key roles in cellular differentiation, proliferation, and homeostasis. As such, NFI family members engage in large number of interactions with other proteins and the chromatin. However, despite their well-established significance, the NFIs interactomes, their dynamics, and their functions have not been comprehensively examined. Here, we employed complementary omics-level techniques, i.e. interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions in different cell lines. Our analyses included all four NFI family members, and a less studied short isoform of NFIB (NFIB4), which lacks the DNA binding domain. We observed that, despite exhibiting redundancy, each family member had unique high-confidence interactors and target genes, suggesting distinct roles within the transcriptional regulatory networks. The study revealed that NFIs interact with other TFs to co-regulate a broad range of regulatory networks and cellular processes. Notably, time-dependent proximity-labeling unveiled a highly dynamic nature of NFI protein-protein interaction networks and hinted at temporal modulation of NFI interactions. Furthermore, gene ontology (GO) enrichment analysis of NFI interactome and targetome revealed the involvement of NFIs in transcriptional regulation, chromatin organization, and cellular signaling pathways, and pathways related with cancer. Additionally, we observed that NFIB4 engages with proteins associated with mRNA regulation, which suggests that NFIs have roles beyond traditional DNA binding and transcriptional modulation. We propose that NFIs may function as potential pioneering TFs, given their role in regulating the DNA binding ability of other TFs and their interactions with key chromatin remodeling complexes, thereby influencing a wide range of cellular processes. These insights into NFI protein-protein interactions and their dynamic, context-dependent nature provide a deeper understanding of gene regulation mechanisms and hint at the role of NFIs as master regulators.

Identification of phosphatases that dephosphorylate the co-chaperone BAG3.

The co-chaperone BAG3 plays critical roles in maintaining cellular proteostasis. It associates with 14-3-3 proteins during the trafficking of aggregation-prone proteins and facilitates their degradation through chaperone-assisted selective autophagy in cooperation with small heat shock proteins. Although reversible phosphorylation regulates BAG3 function, the involved phosphatases remain unknown. Here, we used affinity purification mass spectrometry to identify phosphatases that target BAG3. Of the hits, we evaluated the involvement of protein phosphatase-1 (PP1) using chemical inhibitors and activators in in vitro and cellular approaches. Our results demonstrate that PP1 can dephosphorylate BAG3-pS136 in cells and counteract 14-3-3γ association with BAG3 at this motif. Furthermore, protein phosphatase-5 (PP5) co-enriched with proteostasis-related proteins, and it has the capacity to dephosphorylate a BAG3 phosphorylation-site cluster regulating the interaction of BAG3 with small heat shock proteins and BAG3-mediated protein degradation. Our findings provide new insights into the regulation of BAG3 by phosphatases. This paves the way for future research focused on the precise control of BAG3 function through its regulatory proteins, potentially holding therapeutic promise for diseases characterized by disrupted proteostasis.

Abstract 4134148: Reduced BMPR2 in Monocytes De-represses HERV-K and Sustains Inflammation in Pulmonary Arterial Hypertension

Background&Rationale: Human endogenous retroviral (HERV) elements are embedded in 8% of our genome. They remain quiescent but can be transiently activated by viral infection to induce an interferon (IFN) response. In pulmonary arterial hypertension (PAH), HERV-K expression is elevated in myeloid cells, and thus can promote inflammation observed in pulmonary arteries (PAs) with progressive obstructive remodeling. PAH is linked to a mutation or a decrease in BMPR2 expression which our laboratory has recently shown in monocytes (MONO). HERVs are silenced by SUMOylated (S) TRIM28 (KAP1, a methylase) and SPEN (a histone deacetylase) factors that are also sequestered by the lnc RNA XIST which is increased in females to inactivate the extra X-chromosome. When TRIM28 is phosphorylated (P) by DNA-PK, it loses methylase activity and can act as a transcription factor. Hypothesis: Reduced BMPR2 in MONO results in DNA-PK mediated P-TRIM28 causing demethylation of XIST targets (X-related genes) and HERV-K thus increasing their expression. Competitive SPEN binding to the increased XIST also de-represses HERV-K . Approach: We used THP-1 MONOs with BMPR2 shRNA and induced pluripotent stem cell (i) MONOs derived from BMPR2 mutant PAH patients and assessed HERV-K , XIST , IFNs and related our findings to DNA-PK–P-TRIM28 and sequestration of SPEN. Results: XIST and HERV-K mRNA were significantly elevated in THP-1 MONOs with shBMPR2 vs. shControl and in PAH-iMONO vs. control iMONO based on gender. Reduced BMPR2 also increased IFN genes. These findings were consistent with nuclear DNA-PK and P-TRIM28 shown to bind in affinity purification mass spectrometry analysis. Bisulfite conversion assay demonstrated decreased methylation of HERV-K in BMPR2 -deficient MONOs attributed to a reduction in the methylase S-TRIM28. RNA-IP in THP-1 MONOs with shBMPR2 vs. shControl indicated increased binding of XIST with TRIM28 and SPEN, suggesting a competitive reduction in binding to HERV-K. Conclusions: Loss of BMPR2 increases XIST and HERV-K in MONO related to DNA-PK induced P-TRIM28 and reduced methylase S-TRIM28. Increased HERV-K may also be a function of the increase in XIST sequestering its deacetylase SPEN. These molecular features underlie a chronic HERV-K -IFN inflammatory response in PAH. They may explain the female predisposition to PAH, because the increase in XIST in females heightens the propensity to activate HERV-K -IFN signaling and chronic inflammation in PAs infiltrated by MONO.

EXTH-26. EXPLORING THE ROLE OF ANDROGEN RECEPTOR SIGNALING IN GLIOBLASTOMA IN AN ORTHOTOPIC ANIMAL MODEL

Abstract OBJECTIVE Gliomas, particularly glioblastoma which is the most common and deadly type, frequently express androgen receptors (AR). We systematically characterized AR expression in low- and high-grade gliomas to investigate whether disrupting AR signaling with readily available blood-brain-barrier penetrant drugs could improve treatment. METHODS Low- and high-grade glioma samples were stained for AR by immunohistochemistry, with AR-expression correlated to grade and sex. Localization of AR and the influence of dihydrotestosterone (DHT) and anti-androgens in glioblastoma cell line (U251) were determined by immunofluorescence and immunoblot. Building on previous in vitro data, we used an orthotopic AR-positive patient-derived model (RN1) to test the efficacy of seviteronel, a blood-brain-barrier penetrant anti-androgen. RESULTS Two thirds of biopsy samples were positive for AR expression by IHC (n=25/39). AR expression correlated with increasing tumor grade (p<0.05) but was similar between sexes (p=0.94). Anti-androgens enzalutamide and seviteronel prevented DHT-induced nuclear translocation of AR; however, seviteronel did so to a lesser extent, as measured by immunofluorescence and immunoblot. In the mouse model, seviteronel significantly improved survival compared to vehicle (n=12, 36.0 vs 27.5 days, p=0.03), and demonstrated reduced Ki67 expression in tumors at the endpoint (68.9% vs 85.0%, p<0.001). Combining seviteronel with temozolomide limited tumor growth as measured by bioluminescence imaging, but did not significantly extend survival compared to temozolomide alone (n=16, 63.5 vs 56.5 days, p=0.37). Adding seviteronel to radiotherapy did not extend survival compared to radiotherapy alone (n=16, 33.0 vs 39.0 days, p=0.19). CONCLUSIONS Targeting AR signaling with blood-brain-barrier penetrant anti-androgens may represent a promising biomarker-directed therapeutic strategy. The mechanisms underlying the effects of seviteronel are being further investigated through affinity-purification mass spectrometry analysis and CUT&RUN sequencing of untreated and treated glioma cells. Additional animal experiments are underway to examine the role of biological sex and the glioma immune microenvironment on the efficacy of anti-androgens in vivo.

Recent Advances in Mass Spectrometry-based Protein Interactome Studies

The foundation of all biological processes is the network of diverse and dynamic protein interactions with other molecules in cells known as the interactome. Understanding the interactome is crucial for elucidating molecular mechanisms but has been a longstanding challenge. Recent developments in mass spectrometry (MS)-based techniques, including affinity purification, proximity labeling, cross-linking, and co-fractionation mass spectrometry (MS), have significantly enhanced our abilities to study the interactome. They do so by identifying and quantifying protein interactions, yielding profound insights into protein organizations and functions. This review summarizes recent advances in MS-based interactomics, focusing on the development of techniques that capture protein-protein, protein-metabolite, and protein-nucleic acid interactions. Additionally, we discuss how integrated MS-based approaches have been applied to diverse biological samples, focusing on significant discoveries that have leveraged our understanding of cellular functions. Finally, we highlight state-of-the-art bioinformatic approaches for predictions of interactome and complex modeling, as well as strategies for combining experimental interactome data with computation methods, thereby enhancing the ability of MS-based techniques to identify protein interactomes. Indeed, advances in MS technologies and their integrations with computational biology provide new directions and avenues for interactome research, leveraging new insights into mechanisms that govern the molecular architecture of living cells and, thereby, our comprehension of biological processes.

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

EMC3 regulates trafficking and pulmonary toxicity of the SFTPCI73T mutation associated with interstitial lung disease.

The most common mutation in surfactant protein C gene (SFTPC), SFTPCI73T, causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPCI73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity purification-mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including Valosin Containing Protein (VCP) and its interactors. Treatment of SftpcI73T knock-in mice and SFTPCI73T expressing iAT2 cells derived from SFTPCI73T patient-specific iPSCs with the specific VCP inhibitor CB5083 restored alveolar structure and SFTPCI73T trafficking respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPCI73T mutant, providing novel therapeutical targets for SFTPCI73T-associated interstitial lung disease.

Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics.

Targeted protein degradation and induced proximity refer to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of glues remains challenging, unbiased discovery methods are needed to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue targets. By mapping the targets of 20 CRBN-binding molecular glues, we identify 298 protein targets and demonstrate the utility of enrichment methods for identifying novel targets overlooked using established methods. We use a computational workflow to estimate target confidence and perform a biochemical screen to identify a lead compound for the new non-ZF target PPIL4. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

Hippocampal dipeptidyl peptidase 9 bidirectionally regulates memory associated with synaptic plasticity

IntroductionSubtypes of the dipeptidyl peptidase (DPP) family, such as DPP4, are reportedly associated with memory impairment. DPP9 is widely distributed in cells throughout the body, including the brain. However, whether DPP9 regulates memory has not yet been elucidated. ObjectivesThis study aimed to elucidate the role of DPP9 in memory, as well as the underlying molecular mechanism. MethodsWe performed immunofluorescence on mouse brains to explore the distribution of DPP9 in different brain regions and used AAV vectors to construct knockdown and overexpression models. The effects of changing DPP9 expression on memory were demonstrated through behavioral experiments. Finally, we used electrophysiology, proteomics and affinity purification mass spectrometry (AP-MS) to study the molecular mechanism by which DPP9 affects memory. ResultsHere, we report that DPP9, which is found almost exclusively in neurons, is expressed and has enzyme activity in many brain regions, especially in the hippocampus. Hippocampal DPP9 expression increases after fear memory formation. Fear memory was impaired by DPP9 knockdown and enhanced by DPP9 protein overexpression in the hippocampus. According to subsequent hippocampal proteomics, multiple pathways, including the peptidase pathway, which can be bidirectionally regulated by DPP9. DPP9 directly interacts with its enzymatic substrate neuropeptide Y (NPY) in neurons. Hippocampal long-term potentiation (LTP) is also bidirectionally regulated by DPP9. Moreover, inhibiting DPP enzyme activity impaired both LTP and memory. In addition, AP-MS revealed that DPP9-interacting proteins are involved in the functions of dendritic spines and axons. By combining AP-MS and proteomics, DPP9 was shown to play a role in regulating actin functions. ConclusionTaken together, our findings reveal that DPP9 affects the CNS not only through enzymatic activity but also through protein–protein interactions. This study provides new insights into the molecular mechanisms of memory and DPP family functions.

A protein blueprint of the diatom CO2-fixing organelle

Diatoms are central to the global carbon cycle. At the heart of diatom carbon fixation is an overlooked organelle called the pyrenoid, where concentrated CO2 is delivered to densely packed Rubisco. Diatom pyrenoids fix approximately one-fifth of global CO2, but the protein composition of this organelle is largely unknown. Using fluorescence protein tagging and affinity purification-mass spectrometry, we generate a high-confidence spatially defined protein-protein interaction network for the diatom pyrenoid. Within our pyrenoid interaction network are 10 proteins with previously unknown functions. We show that six of these form a shell that encapsulates the Rubisco matrix and is critical for pyrenoid structural integrity, shape, and function. Although not conserved at a sequence or structural level, the diatom pyrenoid shares some architectural similarities to prokaryotic carboxysomes. Collectively, our results support the convergent evolution of pyrenoids across the two main plastid lineages and uncover a major structural and functional component of global CO2 fixation.

Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.

Molecular chaperones and co-chaperones are highly conserved cellular components that perform a variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an assembly chaperone and serves as a paradigm for studying how specific RNAs are identified and paired with their client substrate proteins to form RNPs. SMN is the eponymous component of a large complex, required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs), that localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN protein forms the oligomeric core of this complex, and missense mutations in the human SMN1 gene are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known. However, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. We carried out affinity purification mass spectrometry (AP-MS) of Drosophila SMN complexes using fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially associated with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Insights into the Role of VPS39 and Its Interaction with CP204L and A137R in ASFV Infection.

The African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal disease in swine, for which no antiviral drugs or vaccines are currently available. Studying viral-host protein-protein interactions advances our understanding of the molecular mechanisms underlying viral replication and pathogenesis and can facilitate the discovery of antiviral therapeutics. In this study, we employed affinity tagging and purification mass spectrometry to characterize the interactome of VPS39, an important cellular factor during the early phase of ASFV replication. The interaction network of VPS39 revealed associations with mitochondrial proteins involved in membrane contact sites formation and cellular respiration. We show that the ASFV proteins CP204L and A137R target VPS39 by interacting with its clathrin heavy-chain functional domain. Furthermore, we elaborate on the potential mechanisms by which VPS39 may contribute to ASFV replication and prioritize interactions for further investigation into mitochondrial protein function in the context of ASFV infection.

Regulation of Drosophila brain development and organ growth by the Minibrain/Rala signaling network.

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer; however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development and organ growth. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Mnb also promotes re-localization of Rlip from the nucleus to the cytoplasm in cultured cells. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in organ growth and neurodevelopment.

PRC1 Protein Subcomplexes Architecture: Focus on the Interplay between Distinct PCGF Subunits in Protein Interaction Networks.

The six PCGF proteins (PCGF1-6) define the biochemical identity of Polycomb repressor complex 1 (PRC1) subcomplexes. While structural and functional studies of PRC1 subcomplexes have revealed their specialized roles in distinct aspects of epigenetic regulation, our understanding of the variation in the protein interaction networks of distinct PCGF subunits in different PRC1 complexes is incomplete. We carried out an affinity purification mass spectrometry (AP-MS) screening of three PCGF subunits, PCGF1 (NSPC1), PCGF2 (MEL18), and PCGF4 (BMI1), to define their interactome and potential cellular function in pluripotent human embryonal carcinoma cell "NT2". The bioinformatic analysis revealed that these interacting proteins cover a range of functional pathways, often involved in cell biology and chromatin regulation. We also found evidence of mutual regulation (at mRNA and protein level) between three distinct PCGF subunits. Furthermore, we confirmed that the disruption of these subunits results in reduced cell proliferation ability. We reveal an interplay between the compositional diversity of the distinct PCGF containing PRC1 complex and the potential role of PCGF proteins within the wider cellular network.

Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39

It has been revealed recently that the RNA-binding motif protein RBM39 is highly expressed in several cancers, which results in poor patient survival. However, how RBM39 is regulated in gastric cancer cells is unknown. Here, affinity purification-mass spectrometry and a biochemical screening are employed to identify the RBM39-interacting proteins and the deubiquitinating enzymes (DUBs) that regulate the RBM39 protein level. Integration of the data obtained from these two approaches uncovers USP39 as the potential DUB that regulates RBM39 stability. Bioinformatic analysis discloses that USP39 is increased in gastric cancer tissues and its elevation shortens the duration of overall survival for gastric cancer patients. Biochemical experiments verify that USP39 and RBM39 interact with each other and highly colocalize in the nucleus. Expression of USP39 elevates while USP39 knockdown attenuates the RBM39 protein level and their interaction regulates this modulation and their colocalization. Mechanistic studies reveal that USP39 reduces the K48-linked polyubiquitin chains on RBM39, thus enhancing its stability and increasing the protein level by preventing its proteasomal degradation. USP39 overexpression promotes while its knockdown attenuates the growth, colony formation, migration, and invasion of gastric cancer cells. Interestingly, overexpression of RBM39 partially restores the impact of USP39 depletion while RBM39 knockdown partially abolishes the effect of USP39 overexpression on the growth, colony formation, migration, and invasion of gastric cancer cells. Collectively, this work identifies the first DUB for RBM39 and elucidates the regulatory functions and the underlying mechanism, providing a possible alternative approach to suppressing RBM39 by inhibiting USP39 in cancer therapy.

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Ferroptosis is a non-apoptotic form of cell death resulting from the iron-dependent accumulation of lipid peroxides. Colorectal cancer (CRC) cells accumulate high levels of intracellular iron and reactive oxygen species (ROS) and are thus particularly sensitive to ferroptosis. The compound (S)-RSL3 ([1S,3R]-RSL3) is a commonly used ferroptosis inducing compound that is currently characterized as a selective inhibitor of the selenocysteine containing enzyme (selenoprotein) Gluathione Peroxidase 4 (GPx4), an enzyme that utilizes glutathione to directly detoxify lipid peroxides. However, through chemical controls utilizing the (R) stereoisomer of RSL3 ([1R,3R]-RSL3) that does not bind GPx4, combined with inducible genetic knockdowns of GPx4 in CRC cell lines, we revealed that GPx4 dependency does not always align with (S)-RSL3 sensitivity, questioning the current characterization of GPx4 as the central regulator of ferroptosis. Utilizing affinity pull-down mass spectrometry with chemically modified (S)-RSL3 probes we discovered that the effects of (S)-RSL3 extend far beyond GPx4 inhibition, revealing that (S)-RSL3 is a broad and non-selective inhibitor of selenoproteins. To further investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed additional chemical and genetic approaches. We found that the selenoprotein inhibitor auranofin, an FDA approved gold-salt, chemically induced oxidative cell death and ferroptosis in both in-vitro and in-vivo models of CRC. Consistent with these data, we found that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translation of selenoproteins, is essential for the in-vitro growth and xenograft survival of CRC cell lines. In summary, these findings recharacterize the mechanism of action of the most commonly used ferroptosis inducing molecule, (S)-RSL3, and reveal that broad inhibition of selenoproteins is a promising novel therapeutic angle for the treatment of CRC.

Affinity purification mass spectrometry characterisation of the interactome of receptor tyrosine kinase proline-rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40 % of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer, RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

Proteostasis Landscapes of Selective versus Poorly Responsive CFTR Variants Reveals Structural Vulnerabilities to Correction.

Cystic Fibrosis (CF) is a lethal genetic disorder caused by variants in CF transmembrane conductance regulator (CFTR). Many disease variants are treatable with corrector compounds, which enhance the folding and trafficking of CFTR. However, correctors fail to elicit a response for every CFTR variant. Approximately 3% of persons with CF harbor poorly responsive CFTR variants. Here, we reveal that a group of poorly responsive variants overlap with selectively responsive variants in a critical domain interface (nucleotide-binding domain 1/intracellular loop 4 - NBD1/ICL4). Affinity purification mass spectrometry proteomics was used to profile the protein homeostasis (proteostasis) changes of CFTR variants during corrector treatment to assess modulated interactions with protein folding and maturation pathways. Responsive variant interactions converged on similar proteostasis pathways during correction. In contrast, poorly responsive variants subtly diverged, revealing a partial restoration of protein quality control surveillance and a capacity to correct some mutations. Computational structural modeling showed that corrector VX-445 failed to confer enough NBD1 stability to poorly responsive variants. NBD1 secondary stabilizing mutations rescued poorly responsive variants, revealing structural vulnerabilities in NBD1 required for treating poor responders. Our study provides a framework for discerning the underlying protein quality control and structural defects of CFTR variants not reached with existing drugs. These insights can help expand therapeutics to all susceptible CFTR variants to enhance personalized medicine efforts.

SMP30 Interacts with ROCK1 to Inhibit HCC Invasiveness by Effecting the Epithelial-Mesenchymal Transition.

The senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases with age, and is closely associated with hepatocellular carcinoma (HCC) development. The primary goal of this study was to examine the mechanistic effect of SMP30 on HCC migration and invasion. Bioinformatic and immunohistochemical approaches were used to examine the expression of SMP30 in HCC tissues and its relationship to patient survival. We investigated the effects of SMP30 expression on HCC cell proliferation, migration, invasion, and cell cycle dynamics. cDNA microarray technology was used to determine the gene expression profile of SK-Hep-1 cells following recombinant SMP30 overexpression to identify genes downstream of SMP30 that regulate HCC cell migration and invasion. We identified SMP30 interacting proteins by affinity purification-mass spectrometry (AP-MS) and co-immunoprecipitation/western blotting (COIP-WB). SMP30 expression was lower in HCC tissues compared with normal liver tissues, and its expression positively correlated with overall survival in HCC patients. Additionally, SMP30 overexpression effectively blocked the migratory and invasive properties of SK-Hep-1 cells, but did not affect either proliferation rates or cell cycle. cDNA microarray results confirmed that many of the differentially expressed genes identified are involved in the process of epithelial-mesenchymal transition (EMT). AP-MS and COIP-WB experiments confirmed that Rho-associated protein kinase 1 (ROCK1) interacts with SMP30 in SK-Hep-1 cells, and ROCK1 is known to intimately regulate the EMT process. SMP30 inhibits HCC metastasis by influencing the expression of EMT-related proteins after interacting with ROCK1.

Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2.

Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leverage TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map the proximal human proteomes of all seven canonical type I IFN signaling cascade members under basal and IFN-stimulated conditions. This uncovers a network of 103 high-confidence proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validates several known constitutive protein assemblies, while also revealing novel stimulus-dependent and -independent associations between key signaling molecules. Functional screening further identifies PJA2 as a negative regulator of IFN signaling via its E3 ubiquitin ligase activity. Mechanistically, PJA2 interacts with TYK2 and JAK1, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2 thereby restraining downstream STAT signaling. Our high-resolution proximal protein landscapes provide global insights into the type I IFN signaling network, and serve as a valuable resource for future exploration of its functional complexities.