Affinity Purification Mass Spectrometry Research Articles

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.

The ubiquitin ligase STUB1 suppresses tumorigenesis of renal cell carcinomas through regulating YTHDF1 stability.

STIP1 homology and U-box protein 1 (STUB1), a crucial member of the RING family E3 ubiquitin ligase, serves dual roles as an oncogene and a tumor suppressor in various human cancers. However, the role and mechanism of STUB1 in clear cell renal cell carcinoma (ccRCC) remain poorly defined. Here, we identified YTHDF1 as a novel STUB1 interaction partner using affinity purification mass spectrometry (AP-MS). Furthermore, we revealed that STUB1 promotes the ubiquitination and degradation of YTHDF1. Consequently, STUB1 depletion leads to YTHDF1 up-regulation in renal cancer cells. Functionally, STUB1 depletion promoted migration and invasion of ccRCC cells in a YTHDF1-dependent manner. Additionally, depletion of STUB1 also increased the tumorigenic potential of ccRCC in a xenograft model. Importantly, STUB1 expression is down-regulated in ccRCC tissues, and its low expression level correlates with advanced tumor stage and poor overall survival in ccRCC patients. Taken together, these findings reveal that STUB1 inhibits the tumorigenicity of ccRCC by regulating YTHDF1 stability.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein–protein and protein–RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein–protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

USP7 interacts with and destabilizes oncoprotein SET

Oncoprotein SE translocation (SET) is frequently overexpressed in different types of tumors and correlated with poor prognosis of cancer patients. Targeting SET has been considered a promising strategy for cancer intervention. However, the mechanisms by which SET is regulated under cellular conditions are largely unknown. Here, by performing a tandem affinity purification-mass spectrometry (TAP-MS), we identify that the ubiquitin-specific protease 7 (USP7) forms a stable protein complex with SET in cancer cells. Further analyses reveal that the acidic domain of SET directly binds USP7 while both catalytic domain and ubiquitin-like (UBL) domains of USP7 are required for SET binding. Knockdown of USP7 has no effect on the mRNA level of SET. However, we surprisingly find that USP7 depletion leads to a dramatic elevation of SET protein levels, suggesting that USP7 plays a key role in destabilizing oncoprotein SET, possibly through an indirect mechanism. To our knowledge, our data report the first deubiquitinase (DUB) that physically associates with oncoprotein SET and imply an unexpected regulatory effect of USP7 on SET stability.

Abstract 7101: Remodeling of cancer cells by chemotherapy

Abstract Cisplatin is a commonly administrated chemotherapy drug for cancer treatment. Although the direct mechanism of cisplatin is largely well-defined, it is challenging to comprehensively assess its systemic effects. Cellular responses to perturbations are intricate processes involving modifications to protein complexes across diverse cellular compartments. Therefore, substantial research endeavors have been directed towards unraveling protein organization in cells. We recently built a multiscale integrated cell map, MuSIC, that combines fluorescence-stained images and interactions derived from affinity purification mass spectrometry to allow comprehensive characterization of cellular structure and functional networks of 5,254 proteins of the human bone osteosarcoma epithelial line (U2-OS). To exploit the biology of U2OS, we integrated dynamics into this map to determine the proteome reorganization upon cisplatin exposure. Thus, we developed a data-driven approach integrating high throughput size exclusion chromatography mass spectrometry (SEC-MS) data with large-scale cell maps built from static-state information to systematically map at all scales the remodeling of cell architecture in drug-treated cells. Here, we acquired drug-perturbed protein-protein interactions (PPIs) from the SEC-MS data by treating U2-OS with cisplatin. Integrating the differential interactions identified from SEC-MS PPIs with the scaffold large-scale hierarchy (MuSIC) allows retention of the upper cellular organization to systematically map drug impact on protein complexes in the cell. This pipeline serves as an explorative method to identify the drug-responsive organelles, alterations of hierarchical structures, and translocation of proteins. Apart from the well known alterations of DNA damage repair complexes, we also recapitulate remodeling in metabolism pathways, splicing, cAMP-dependent kinase activity, and histone modification. We identify 26 novel protein assemblies containing members that are previously associated with cisplatin biology. This pipeline exploits the potential of a well-established network by integrating the high throughput data modality, allowing for the screening of a range of chemical agents and stimuli to generate differential networks. Citation Format: Gege Qian, Leah Schaffer, Kyung-Mee Moon, Leonard Foster, Trey Ideker, Mengzhou Hu, Sahar Alkhairy, Emma Lundberg. Remodeling of cancer cells by chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7101.

Abstract 5591: ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer

Abstract The SWI/SNF chromatin remodeler utilizes energy of ATP hydrolysis to slide or evict nucleosomes or histones, respectively, thus enabling nuclear processes by driving an ‘open’ chromatin architecture. The mammalian SWI/SNF, also termed the BrG1/Brm associated factor (BAF) complex, is the major chromatin remodeler in ontogeny and adult life. Three major BAF complexes namely canonical (cBAF), polybromo (pBAF), and non-canonical (ncBAF) have been studied. BAF components, classified as a tumor suppressors, exhibit frequent mutations in cancers. Though, studies have evaluated functional roles of BAF subunits, very few attempted to study BAF mutations identified across cancer types. In our previous studies, we identified tumor specific mutations in the gene encoding ‘A-T rich interaction domain containing 1B’ (ARID1B), within its nuclear localization signal (NLS). NLS-inactivated ARID1B exhibited cytoplasmic localization and promoted tumorigenesis by activating the ERK and Wnt/β-catenin pathways. More recently, we detected inactivating mutations in the gene encoding ARID2, a component of the pBAF complex and an ARID1B paralogue. We now present detailed characterization of tumor-specific ARID2 truncations that were presumed to be inactivating in nature. One such truncation mutation viz. p.Ser989fsArg21*, identified in our previous study, is expected to yield an ARID2 protein truncating at amino acid position 1010 (as against the full length of 1835 amino acids). Scrutiny of The Cancer Genome Atlas cBioPortal and Cancer Cell Line Encyclopedia mutation data bases revealed several ARID2 mutations expected to truncate the protein around the 1010 position. Surprisingly, ectopic expression of truncated ARID2 forms promoted oncogenic function in both ARID2 proficient and deficient backgrounds, thus validating a neomorphic gain of function for the truncated protein. Fluorescence based intracellular localization assays revealed a surprising cytoplasmic localization of truncated ARID2. Further, we mapped the ARID2 NLS by in silico analysis followed by evaluation of several deletion constructs, to be located at 1488-1518 position; thus, explaining the cytoplasmic localization of ARID2 forms truncating around 1010 position. Further, truncated ARID2 was unable to bind other pBAF components and did not sequester other pBAF components to the cytoplasm, in both ARID2 proficient and deficient cells. Tandem Affinity Purification Mass-spectrometry revealed putative interaction partners for truncated ARID2; analysis is currently underway to reveal the mechanism of tumorigenic induction exhibited by truncated ARID2. Immunohistochemistry on colorectal and breast cancer tissue microarrays revealed ARID2 cytoplasmic localization in a significant fraction of samples. We have discovered a novel YIN-YANG function of BAF components expected to yield efficient therapeutic options in cancer. Citation Format: Sanjana Sarkar, Murali Dharan Bashyam. ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5591.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.MethodsSingle-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.Data availabilityThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.Graphical

LRRC59 promotes the progression of oral squamous cell carcinoma by interacting with SRP pathway components and enhancing the secretion of CKAP4-containing exosomes

BackgroundAs a ribosome receptor, LRRC59 was thought to regulate mRNA translation on the ER membrane. Evidence suggests that LRRC59 is overexpressed in a number of human malignancies and is associated with poor prognoses, but its primary biological function in the development of oral squamous cell carcinoma (OSCC) remains obscure. ObjectiveThe purpose of this study is to investigate at the expression changes and functional role of LRRC59 in OSCC. MethodsLRRC59 gene expression and correlation with prognosis of OSCC patients were first examined using the data from The Cancer Genome Atlas (TCGA) databases. Following that, a series of functional experiments, including cell counting kit-8, cell cycle analysis, wound healing assays, and transwell assays, were carried out to analyze the biological roles of LRRC59 in tumor cells. Mechanistically, we employed Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach to isolate and identify protein complexes of LRRC59. Downstream regulatory proteins of LRRC59 were verified through immunoprecipitation and immunofluorescence experiments. Furthermore, we isolated exosomes from OSCC cell supernatant and conducted co-culture experiments to examine the effect of LRRC59 knockdown on OSCC cells. ResultsIn samples from OSCC patients, LRRC59 was highly expressed and correlated with poor prognoses. Moreover, the gene sets analysis based on TCGA RNA-seq data indicated that LRRC59 seemed to be strongly related with protein secretory and OSCC migration. Upregulated levels of LRRC59 are more prone to lymph node metastasis in OSCC patients. LRRC59 knockdown impaired the ability of OSCC cell proliferation, migration, and invasion invitro. Mechanistically, our TAP-MS data situate LRRC59 in a functional nexus for mRNA translation regulation via interactions with SRP pathway components, translational initiation factors, CRD-mediated mRNA stabilization factors. More importantly, we found that LRRC59 interacted with cytoskeleton-associated protein 4 (CKAP4) and promoted the formation of CKAP4-containing exosomes. We also revealed that the LRRC59-CKAP4 axis was a crucial regulator of CKAP4-containing exosome secretion in OSCC cells for migration and invasion. ConclusionsTherefore, based on our findings, LRRC59 may serve as a potential biomarker for OSCC patients, and LRRC59-induced exosome secretion via the CKAP4 axis may serve as a potential therapeutic target for OSCC.

Phage Display Identified Novel Leydig Cell Homing Peptides for Testicular Targeting.

Conventional drug delivery methods to treat testicular disorders face various challenges, which could be circumvented by using targeted drug delivery. Testicular cell targeting ligands, such as Leydig cell homing peptides, would be an excellent choice to achieve the targeted delivery of drugs to the testis. In this study, Leydig cell homing peptides (LCHPs), LCHP1 and LCHP2, were identified via in vitro, followed by in vivo biopanning of a phage display peptide library and next-generation sequencing. Both of the LCHPs were validated in vitro for their specific Leydig cell and in vivo testis targeting potential. Furthermore, molecular targets of the LCHP1 and LCHP2 were identified using affinity purification mass spectrometry (APMS). The LCHP1 and LCHP2 are able to specifically target Leydig cells of the testis and undergo cell internalization as well as target the testis at the in vivo level, hence providing an opportunity to be utilized as a potential ligand for drug delivery to the testis.

Micropillar arrays, wide window acquisition and AI-based data analysis improve comprehensiveness in multiple proteomic applications.

Comprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited proteomic coverage and dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition and the AI-based CHIMERYS search engine to achieve excellent proteomic comprehensiveness for bulk proteomics, affinity purification mass spectrometry and single cell proteomics. Our data show that µPACs identify ≤50% more peptides and ≤24% more proteins, while offering improved throughput, which is critical for large (clinical) proteomics studies. Combining wide precursor isolation widths of m/z 4-12 with the CHIMERYS search engine identified +51-74% and +59-150% more proteins and peptides, respectively, for single cell, co-immunoprecipitation, and multi-species samples over a conventional workflow at well-controlled false discovery rates. The workflow further offers excellent precision, with CVs <7% for low input bulk samples, and accuracy, with deviations <10% from expected fold changes for regular abundance two-proteome mixes. Compared to a conventional workflow, our entire optimized platform discovered 92% more potential interactors in a protein-protein interaction study on the chromatin remodeler Smarca5/Snf2h. These include previously described Smarca5 binding partners and undescribed ones including Arid1a, another chromatin remodeler with key roles in neurodevelopmental and malignant disorders.

Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

Nucleolin binds to and regulates transcription of hepatitis B virus covalently closed circular DNA minichromosome.

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.

Co-fractionation-mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain.

Human mitochondrial (mt) protein assemblies are vital for neuronal and brain function, and their alteration contributes to many human disorders, e.g., neurodegenerative diseases resulting from abnormal protein-protein interactions (PPIs). Knowledge of the composition of mt protein complexes is, however, still limited. Affinity purification mass spectrometry (MS) and proximity-dependent biotinylation MS have defined protein partners of some mt proteins, but are too technically challenging and laborious to be practical for analyzing large numbers of samples at the proteome level, e.g., for the study of neuronal or brain-specific mt assemblies, as well as altered mtPPIs on a proteome-wide scale for a disease of interest in brain regions, disease tissues or neurons derived from patients. To address this challenge, we adapted a co-fractionation-MS platform to survey native mt assemblies in adult mouse brain and in human NTERA-2 embryonal carcinoma stem cells or differentiated neuronal-like cells. The workflow consists of orthogonal separations of mt extracts isolated from chemically cross-linked samples to stabilize PPIs, data-dependent acquisition MS to identify co-eluted mt protein profiles from collected fractions and a computational scoring pipeline to predict mtPPIs, followed by network partitioning to define complexes linked to mt functions as well as those essential for neuronal and brain physiological homeostasis. We developed an R/CRAN software package, Macromolecular Assemblies from Co-elution Profiles for automated scoring of co-fractionation-MS data to define complexes from mtPPI networks. Presently, the co-fractionation-MS procedure takes 1.5-3.5 d of proteomic sample preparation, 31 d of MS data acquisition and 8.5 d of data analyses to produce meaningful biological insights.

Abstract 17551: Mapping the Interactome of FLNC in Restrictive Cardiomyopathy

Introduction: Filamin C (FLNC) is an actin crosslinking protein that organizes structural components of the sarcomere and signal transduction complexes. Human mutations in FLNC have been linked to dilated, hypertrophic and restrictive cardiomyopathies. The mechanism connecting mutations in FLNC to sarcomeric impairment remains undefined. We recently reported the ability to model FLNC cardiomyopathy using induced pluripotent stem cell (iPSC) derived engineered heart tissue. Here, we employ a knock-in approach to epitope tag endogenous alleles in iPSC derived cardiomyocytes to explore FLNC genotype-interactome relationships using affinity purification mass spectrometry. Methods: An iPSC line was generated from a patient with restrictive cardiomyopathy caused by an insertion deletion mutation in the ROD2 domain FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp). CRISPR-Cas9 was used to establish a corrected cell line as a wild type control. 3X-Flag motif was introduced to the N terminus of FLNC using CRISPR-Cas9. iPSCs were then differentiated into cardiomyocytes using standard techniques. Affinity purification mass spectrometry (AP-MS) was performed using the Flag epitope. Interaction data was analyzed using Perseus software. Gene ontology (GO) enrichment analysis was performed using ToppFun. Results: 3X-Flag insertion was confirmed using genomic sequencing. AP-MS identified 281 high confidence interactors of mutant FLNC and 208 interactors of wild type FLNC (FDR 0.01, log2FC &gt;1.5) when compared to IgG controls. Interestingly, 161 proteins were shared between the two genotypes. These were enriched in mitochondrial ribosomal proteins and phosphorylase kinase subunits, potentially revealing an unknown role of FLNC in mitochondria translation and metabolism. Thirty-three proteins were significantly differentially bound to mutant FLNC protein, including tropomyosin 4, caldesmon 1, and filamin A (FDR &lt;0.05, log2FC&gt;1). GO analysis of these proteins revealed enrichment of actin cytoskeleton elements, muscle contraction, and muscle system processes. Conclusions: Human mutations in FLNC lead to disruption of key protein interactions, revealing a potential mechanism for disease pathogenesis in FLNC cardiomyopathy.

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells show hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi-enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.

HIC1 interacts with FOXP3 multi protein complex: Novel pleiotropic mechanisms to regulate human regulatory T cell differentiation and function

Transcriptional repressor, hypermethylated in cancer 1 (HIC1) participates in a range of important biological processes, such as tumor repression, immune suppression, embryonic development and epigenetic gene regulation. Further to these, we previously demonstrated that HIC1 provides a significant contribution to the function and development of regulatory T (Treg) cells. However, the mechanism by which it regulates these processes was not apparent. To address this question, we used affinity-purification mass spectrometry to characterize the HIC1 interactome in human Treg cells. Altogether 61 high-confidence interactors were identified, including IKZF3, which is a key transcription factor in the development of Treg cells. The biological processes associated with these interacting proteins include protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism. The results revealed that HIC1 is part of a FOXP3-RUNX1-CBFB protein complex that regulates Treg signature genes thus improving our understanding of HIC1 function during early Treg cell differentiation.