Specific Interactors Research Articles

Interrogating data-independent acquisition LC–MS/MS for affinity proteomics

AbstractData-Independent Acquisition (DIA) LC–MS/MS is an attractive partner for co-immunoprecipitation (co-IP) and affinity proteomics in general. Reducing the variability of quantitation by DIA could increase the statistical contrast for detecting specific interactors versus what has been achieved in Data-Dependent Acquisition (DDA). By interrogating affinity proteomes featuring both DDA and DIA experiments, we sought to evaluate the spectral libraries, the missingness of protein quantity tables, and the CV of protein quantities in six studies representing three different instrument manufacturers. We examined four contemporary bioinformatics workflows for DIA: FragPipe, DIA-NN, Spectronaut, and MaxQuant. We determined that (1) identifying spectral libraries directly from DIA experiments works well enough that separate DDA experiments do not produce larger spectral libraries when given equivalent instrument time; (2) experiments involving mock pull-downs or IgG controls may feature such indistinct signals that contemporary software will struggle to quantify them; (3) measured CV values were well controlled by Spectronaut and DIA-NN (and FragPipe, which implements DIA-NN for the quantitation step); and (4) when FragPipe builds spectral libraries and quantifies proteins from DIA experiments rather than performing both operations in DDA experiments, the DIA route results in a larger number of proteins quantified without missing values as well as lower CV for measured protein quantities.

A SeNA-based study of in-service teachers’ interaction patterns in an online learning community

In-service teachers, constrained by work commitments, adopt online learning methods for their graduate studies, emphasizing the significance of exploring the construction of online learning communities. Analyzing collaborative conversation texts within the online learning process of the “Learning Science” course at a university in Beijing, this study employed social epistemic network analysis (SeNA) to investigate interaction patterns of in-service teachers and propose optimization strategies. The findings of this study show that in asynchronous discussions without intervention, the overall situation of collaborative conversations among in-service teachers tends to be low-level. The social interaction network shows polycentricity, categorizing members into core and peripheral interactors based on distinct social interaction characteristics, highlighting substantial disparities in epistemic networks. Core interactors prioritize engaging with others, yet their contributions primarily echo interactions, reflecting superficial knowledge dimensions. Conversely, specific peripheral interactors prioritize independent reflection, sharing content largely derived from personal experiences or knowledge, signifying a commitment to independent and in-depth thinking within deeper knowledge dimensions. Moreover, learners’ online interactions are influenced by individual characteristics, instructional evaluation and the learning community atmosphere.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM)

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins.In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart.To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

SPTBN1 Mediates the Cytoplasmic Constraint of PTTG1, Impairing Its Oncogenic Activity in Human Seminoma.

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.

Interactions of Histone Deacetylase 6 with DNA Damage Repair Factors Strengthen its Utility as a Combination Drug Target in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy in women. The low survival rate is largely due to drug resistance. Approximately 80% of patients who initially respond to treatment relapse and become drug-resistant. The lack of effective second-line therapeutics remains a substantial challenge for BRCA-1/2 wild-type HGSOC patients. Histone Deacetylases (HDACs) are promising targets in HGSOC treatment; however, the mechanism and efficacy of HDAC inhibitors are understudied in HGSOC. In order to consider HDACs as a treatment target, an improved understanding of their function within HGSOC is required. This includes elucidating HDAC6-specific protein-protein interactions. In this study, we carried out substrate trapping followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate HDAC6 catalytic domain (CD)-specific interactors in the context of BRCA-1/2 wild-type HGSOC. Overall, this study identified new HDAC6 substrates that may be unique to HGSOC. The HDAC6-CD1 mutant condition contained the largest number of significant proteins compared to the CD2 mutant and the CD1/2 mutant conditions, suggesting the HDAC6-CD1 domain has catalytic activity that is independent of CD2. Among the identified substrates were proteins involved in DNA damage repair including PARP proteins. These findings further justify the use of HDAC inhibitors as a combination treatment with platinum chemotherapy agents and PARP inhibitors in HGSOC.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gβγ simultaneously, mimicking testosterone actions, as well as for specific Gβγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαβγ and seven Gβγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gβγ and Gαβγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model.

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments.In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum.Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and post-symptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACSpatients.

Abstract B029: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of the long RAP1GDS1 isoform. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of the long RAP1GDS1 isoform, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Eric Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B029.

Abstract 3920: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of RAP1GDS1-607. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of RAP1GDS1-607, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. In conclusion, these data demonstrate a specific role of the long RAP1GDS1 isoform in the membrane-localization and activation of KRAS, which supports its role as a potential therapeutic target for KRAS mutant tumors. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3920.

Tissue specific LRRK2 interactomes reveal a distinct striatal functional unit.

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease. Despite substantial research efforts, the physiological and pathological role of this multidomain protein remains poorly defined. In this study, we used a systematic approach to construct the general protein-protein interactome around LRRK2, which was then evaluated taking into consideration the differential expression patterns and the co-expression behaviours of the LRRK2 interactors in 15 different healthy tissue types. The LRRK2 interactors exhibited distinct expression features in the brain as compared to the peripheral tissues analysed. Moreover, a high degree of similarity was found for the LRRK2 interactors in putamen, caudate and nucleus accumbens, thus defining a potential LRRK2 functional cluster within the striatum. The general LRRK2 interactome paired with the expression profiles of its members constitutes a powerful tool to generate tissue-specific LRRK2 interactomes. We exemplified the generation of the tissue-specific LRRK2 interactomes and explored the functions highlighted by the "core LRRK2 interactors" in the striatum in comparison with the cerebellum. Finally, we illustrated how the LRRK2 general interactome reported in this manuscript paired with the expression profiles can be used to trace the relationship between LRRK2 and specific interactors of interest, here focusing on the LRRK2 interactors belonging to the Rab protein family.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) co-immunoprecipitated using a time course of infected buffalo sera.

Widespread Fasciola gigantica infection in buffaloes has caused great economic losses in buffalo farming. Studies on F. gigantica excretory and secretory products (FgESP) have highlighted their importance in F. gigantica parasitism and their potential in vaccine development. Identifying FgESP components involved in F. gigantica-buffalo interactions during different periods is important for developing effective strategies against fasciolosis. Buffaloes were assigned to non-infection (n = 3, as control group) and infection (n = 3) groups. The infection group was orally administrated 250 metacercariae. Sera were collected at 3, 10, and 16 weeks post-infection (wpi) for the non-infection group and at 0 (pre-infection), 1, 3, 6, 8, 10, 13, and 16 wpi for the infection group. FgESP components interacting with sera from the non-infection and infection groups assay were pulled down by co-IP and identified using LC-MS/MS. Interacting FgESP components in infection group were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway and gene ontology (GO) functional annotation to infer their potential functions. Proteins of FgESP components identified in the non-infection group at 3, 10, and 16 wpi accounted for 80.5%, 84.3%, and 82.1% of all proteins identified in these three time points, respectively, indicating surroundings did not affect buffalo immune response during maintenance. Four hundred and ninety proteins were identified in the infection group, of which 87 were consistently identified at 7 time points. Following GO analysis showed that most of these 87 proteins were in biological processes, while KEGG analysis showed they mainly functioned in metabolism and cellular processing, some of which were thought to functions throughout the infection process. The numbers of specific interactors identified for each week were 1 (n = 12), 3 (n = 5), 6 (n = 8), 8 (n = 15), 10 (n = 23), 13 (n = 22), and 16 (n = 14) wpi, some of which were thought to functions in specific infection process. This study screened the antigenic targets in FgESP during a dense time course over a long period. These findings may enhance the understanding of molecular F. gigantica-buffalo interactions and help identify new potential vaccine and drug target candidates.

Protein Interaction Networks of Catalytically Active and Catalytically Inactive PqsE in Pseudomonas aeruginosa.

ABSTRACTPseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor RhlR, an interaction that enhances the affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, the virulence function of PqsE can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing enhanced green fluorescent protein-PqsE fusions to define the protein interactomes of wild-type PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wild-type PqsE while not forming associations with PqsE(D73A). In the ΔrhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase for RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing.

Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes.

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to “trap” substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.

LncRNA RUS shapes the gene expression program towards neurogenesis.

The evolution of brain complexity correlates with an increased expression of long, noncoding (lnc) RNAs in neural tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during brain development. We present a first functional characterization of the lncRNA LINC01322, which we term RUS for "RNA upstream of Slitrk3." The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation resulting in arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner.

Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry.

Chemical modification of histone proteins by methylation plays a central role in chromatin regulation by recruiting epigenetic "readers" via specialized binding domains. Depending on the degree of methylation, the exact modified amino acid, and the associated reader proteins histone methylations are involved in the regulation of all DNA-based processes, such as transcription, DNA replication, and DNA repair. Here we present methods to identify histone methylation readers using a mass spectrometry-linked nucleosome affinity purification approach. We provide detailed protocols for the generation of semisynthetic methylated histones, their assembly into biotinylated nucleosomes, and the identification of methylation-specific nucleosome-interacting proteins from nuclear extracts via nucleosome pull-downs and label-free quantitative proteomics. Due to their versatility, these protocols allow the identification of readers of various histone methylations, and can also be adapted to different cell types and tissues, and other types of modifications.

Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex.

Background:Huntington’s disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein.Objective:Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington’s disease pathology.Methods:Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS).Results:We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2.Conclusion:Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington’s disease pathology.

Molecular Characterization of NDL1-AGB1 Mediated Salt Stress Signaling: Further Exploration of the Role of NDL1 Interacting Partners.

Salt stress is considered to be the most severe abiotic stress. High soil salinity leads to osmotic and ionic toxicity, resulting in reduced plant growth and crop production. The role of G-proteins during salt stresses is well established. AGB1, a G-protein subunit, not only plays an important role during regulation of Na+ fluxes in roots, but is also involved in the translocation of Na+ from roots to shoots. N-Myc Downregulated like 1 (NDL1) is an interacting partner of G protein βγ subunits and C-4 domain of RGS1 in Arabidopsis. Our recent in-planta expression analysis of NDL1 reported changes in patterns during salt stress. Based on these expression profiles, we have carried out functional characterization of the AGB1-NDL1 module during salinity stress. Using various available mutant and overexpression lines of NDL1 and AGB1, we found that NDL1 acts as a negative regulator during salt stress response at the seedling stage, an opposite response to that of AGB1. On the other hand, during the germination phase of the plant, this role is reversed, indicating developmental and tissue specific regulation. To elucidate the mechanism of the AGB1-NDL1 module, we investigated the possible role of the three NDL1 stress specific interactors, namely ANNAT1, SLT1, and IDH-V, using yeast as a model. The present study revealed that NDL1 acts as a modulator of salt stress response, wherein it can have both positive as well as negative functions during salinity stress. Our findings suggest that the NDL1 mediated stress response depends on its developmental stage-specific expression patterns as well as the differential presence and interaction of the stress-specific interactors.

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.

Structure‐Function Studies of MUNC LncRNA Reveal Domains Required for Its Promyogenic Phenotype

Although thousands of long non-coding RNAs (lncRNAs) have been already discovered, only few are functionally characterized and even fewer provide insights into molecular mechanism based on their structure. Therefore, studies of lncRNA structure and subsequent elucidation of its function and the regulatory mechanisms are urgently needed. MUNC lncRNA is upregulated in murine skeletal muscles and was firstly described as a facilitator of Myod1 function in myogenesis. There are two MUNC isoforms: spliced and genomic, and both have promyogenic abilities. siMUNC reduces myoblast differentiation, and stable overexpression stimulates promyogenic RNAs. MUNC is also one of the conserved lncRNA and its homolog can be found in human muscles. In this study we focus on the relationship between MUNC lncRNA structure and function. We determined the secondary structure of MUNC and found that specific structural domains are critical in the process of skeletal muscle differentiation. Moreover, we characterized structural features facilitate MUNC binding to specific genomic sites and protein interactors. To analyze the functional similarities and differences between the MUNC two isoforms, we performed RNA-seq on myoblasts overexpressing MUNC spliced or genomic in proliferation and differentiation conditions. We show that both MUNC transcripts can regulate different sets of genes that are important for muscle structure development, RNA processing and apoptosis signaling. We obtained the secondary structure using SHAPE-MaP and we found that MUNC spliced and genomic have six common structural domains and eight common proteins protected sites. Knowing that both MUNC isoforms induce the same promyogenic genes, we systematically deleted the most structurally similar regions. We have identified domains which are required for induction of Myod1, Myogenin and Myh3. We have also found out a domain which is necessary for Myod1 stimulation, but not Myogenin, proving again that MUNC can act both in cis and in trans. Using the data about MUNC functional domains, protein binding partners and genomic sites interactions we can now better characterize this lncRNA and describe its mechanism of function.

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.