TRIM24 directs replicative stress responses to maintain ALT telomeres via chromatin signaling.

An inability to replicate the genome can cause replication stress and genome instability. Here, we develop BLOCK-ID, a proteomic method to identify and visualize proteins at stressed replication forks. This approach successfully identified novel mediators of the replication stress response, including the chromatin acetylation reader protein TRIM24. In validating TRIM24 function, we uncovered its crucial role in coordinating Alternative Lengthening of Telomeres (ALT), a cancer-specific telomere extension pathway involving replication stress. Our data reveal that TRIM24 is directed to telomeres via a p300/CBP-dependent acetylation chromatin signaling cascade, where it organizes ALT-associated PML bodies (APBs) to promote telomere DNA synthesis. Strikingly, we demonstrate that when artificially tethered at telomeres, TRIM24 can stimulate new telomere DNA synthesis in a SUMO-dependent manner, independently of p300/CBP or PML-dependent APBs. Thus, this study identifies a TRIM24 chromatin signaling pathway required for ALT telomere maintenance.

Tandem Mass Tag LC-MS/MS of Aqueous Humor From Individuals With Type 2 Diabetes Without Retinopathy Reveals Early Dysregulation of Synaptic Proteins.

Purpose: An early neurodegenerative component of diabetic retinal disease (DRD) that precedes the vascular findings of clinically diagnosed diabetic retinopathy (DR) is increasingly being recognized. However, the relevant molecular mechanisms and biomarkers for early DRD are poorly defined. The purpose of this study was to uncover novel potential mediators of early diabetic retinal neuronal dysfunction through analysis of the aqueous fluid proteome in preclinical DR. Methods: Aqueous fluid was collected from subjects with type 2 diabetes mellitus (DM) but no clinical DR and from nondiabetic controls undergoing routine cataract surgery. Preoperative spectral-domain optical coherence tomography of the macula was obtained. Tandem mass tag LC-MS/MS was performed to identify proteins differentially present in diabetic and control aqueous fluid, and proteins with >50% change and P < 0.05 were considered significant. Selected results were validated with western blot of human aqueous fluid samples. Results: We identified decreased levels of proteins implicated in neuronal synapse formation and increased levels of inflammatory proteins in the aqueous fluid from patients with type 2 DM but no DR compared with controls. Of the differentially present synaptic proteins that we identified and confirmed with western blot, the majority have not previously been linked with DRD. Conclusions: The proteomic profile of aqueous fluid from individuals with type 2 DM but no DR suggests that retinal neuronal dysfunction and inflammation represent very early events in the pathophysiology of DRD. These findings support the concept that diabetic retinal neurodegeneration precedes vascular pathology and reveal novel potential mediators and/or biomarkers warranting further investigation.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis.

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and hypothesized to drive the loss of immune tolerance to self-antigens in autoimmunity. Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines. Here, we investigate the hypothesis that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage. We show that citrullination alters processing and presentation of autoantigens, resulting in the generation of a unique citrullination-dependent repertoire composed primarily of native sequences. This repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than controls. The generation of this unique repertoire is achieved through altered protease cleavage and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA.

c-Abl Regulates the Pathological Deposition of TDP-43 via Tyrosine 43 Phosphorylation.

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy.

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.

Plasmodium sporozoite phospholipid scramblase interacts with mammalian carbamoyl-phosphate synthetase 1 to infect hepatocytes.

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate.

Examining the effects of cigarette smoke on mouse lens through a multi OMIC approach.

Here, we report a multi OMIC (transcriptome, proteome, and metabolome) approach to investigate molecular changes in lens fiber cells (FC) of mice exposed to cigarette smoke (CS). Pregnant mice were placed in a whole-body smoke chamber and a few days later pups were born, which were exposed to CS for 5 hours/day, 5 days/week for a total of 3½ months. We examined the mice exposed to CS for CS-related cataractogenesis after completion of the CS exposure but no cataracts were observed. Lenses of CS-exposed and age-matched, untreated control mice were extracted and lens FC were subjected to multi OMIC profiling. We identified 348 genes, 130 proteins, and 14 metabolites exhibiting significant (p < 0.05) differential levels in lens FC of mice exposed to CS, corresponding to 3.6%, 4.3%, and 5.0% of the total genes, protein, and metabolites, respectively identified in this study. Our multi OMIC approach confirmed that only a small fraction of the transcriptome, the proteome, and the metabolome was perturbed in the lens FC of mice exposed to CS, which suggests that exposure of CS had a minimal effect on the mouse lens. It is worth noting that while our results confirm that CS exposure does not have a substantial impact on the molecular landscape of the mouse lens FC, we cannot rule out that CS exposure for longer durations and/or in combination with other morbidities or environmental factors would have a more robust effect and/or result in cataractogenesis.

Evaluation of the Sensitivity and Reproducibility of Targeted Proteomic Analysis Using Data-Independent Acquisition for Serum and Cerebrospinal Fluid Proteins.

There is a need for targeted analysis of biological fluids for diagnosis, prognosis, or monitoring the progression of diseases. Cerebrospinal fluid (CSF) and serum have been widely used for the development of protein analysis for neurodegenerative diseases and other diseases, respectively. Recently, data-independent acquisition (DIA) mass spectrometry (MS) has been developed to increase the throughput over data-dependent acquisition (DDA) on screening of a large number of samples and discovery of candidate targets. When it comes to target validation, the analytical performance of targeted analysis is critical. However, the inter- and intralaboratory analytical performances of the DIA-MS for targeted proteomic analysis of CSF and serum samples have not yet been investigated. In this study, we showed that the DIA-MS approach allowed us to identify and quantify 1732 CSF and 424 serum proteins, with 90% of proteins identified and quantified in at least 50% of DIA-MS runs. To evaluate the sensitivity, linearity, and dynamic range of the DIA approach, we included the stable isotope-labeled (SI) peptides into CSF and serum samples with serial dilutions. The lower limit of quantification (LLOQ) of peptides was 0.1-0.5 fmol, and the dynamic range was over 3.53 orders of magnitude, with excellent linearity (r2 < 0.978) in CSF and serum samples. Finally, the reproducibility of the DIA-MS approach was evaluated using entire proteins identified in CSF and serum samples. The intralaboratory three replicate results showed reliable reproducibility with 12.5 and 17.3% of the median coefficient of variation (CV) in both CSF and serum matrices, whereas the median CVs of interlaboratory three replicates were 23.8 and 32.0% in CSF and serum samples, respectively. The comparison of the quantitative result between replicates showed close similarity at intra- and interlaboratories with a median Pearson correlation value of >0.98 in CSF and serum, respectively. In conclusion, we demonstrate the capability of the DIA approach as a targeted proteomic analysis for candidate proteins from CSF and serum samples.

Cigarette Smoke Triggers Loss of Corneal Endothelial Cells and Disruption of Descemet's Membrane Proteins in Mice.

Purpose: To investigate changes at a molecular level in the mouse corneal endothelium (CE) exposed to chronic cigarette smoke (CS). Methods: Pregnant mice (gestation days 18-20) were placed in a whole-body exposure smoking chamber, and a few days later pups were born. After 3.5 months of CS exposure, a ConfoScan4 scanning microscope was used to examine the corneal endothelial cells (CECs) of CS-exposed and control (Ct) mice. The CE was peeled under a microscope and maintained as four biological replicates (two male and two female) for CS-exposed and Ct mice; each replicate consisted of 16 CEs. The proteome of the CE was investigated through mass spectrometry. Results: The CE images of CS-exposed and Ct mice revealed a difference in the shape of CECs accompanied by a nearly 10% decrease in CEC density (P < 0.00003) following CS exposure. Proteome profiling identified a total of 524 proteins exhibiting statistically significant changes in CE from CS-exposed mice. Importantly, proteins associated with Descemet's membrane (DM), including COL4α1, COL4α2, COL4α3, COL4α4, COL4α5, COL4α6, COL8α1, COL8α2, and FN1, among others, exhibited diminished protein levels in the CE of CS-exposed mice. Conclusions: Our data confirm that exposure to CS results in reduced CEC density accompanied by diminished levels of multiple collagen and extracellular matrix proteins associated with DM.

C9orf72 regulates energy homeostasis by stabilizing mitochondrial complex I assembly.

The haploinsufficiency of C9orf72 is implicated in the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the full spectrum of C9orf72 functions remains to be established. Here, we report that C9orf72 is a mitochondrial inner-membrane-associated protein regulating cellular energy homeostasis via its critical role in the control of oxidative phosphorylation (OXPHOS). The translocation of C9orf72 from the cytosol to the inter-membrane space is mediated by the redox-sensitive AIFM1/CHCHD4 pathway. In mitochondria, C9orf72 specifically stabilizes translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1), a crucial factor for the assembly of OXPHOS complex I. C9orf72 directly recruits the prohibitin complex to inhibit the m-AAA protease-dependent degradation of TIMMDC1. The mitochondrial complex I function is impaired in C9orf72-linked ALS/FTD patient-derived neurons. These results reveal a previously unknown function of C9orf72 in mitochondria and suggest that defective energy metabolism may underlie the pathogenesis of relevant diseases.

Persistently Elevated mTOR Complex 1-S6 Kinase 1 Disrupts DARPP-32-Dependent D1 Dopamine Receptor Signaling and Behaviors.

BACKGROUND: The serine-threonine kinase mTORC1 (mechanistic target of rapamycin complex 1) is essential for normal cell function but is aberrantly activated in the brain in both genetic-developmental and sporadic diseases and is associated with a spectrum of neuropsychiatric symptoms. The underlying molecular mechanisms of cognitive and neuropsychiatric symptoms remain controversial. METHODS: The present study examines behaviors in transgenic models that express Rheb, the most proximal known activator of mTORC1, and profiles striatal phosphoproteomics in a model with persistently elevated mTORC1 signaling. Biochemistry, immunohistochemistry, electrophysiology, and behavior approaches are used to examine the impact of persistently elevated mTORC1 on D1 dopamine receptor (D1R) signaling. The effect of persistently elevated mTORC1 was confirmed using D1-Cre to elevate mTORC1 activity in D1R neurons. RESULTS: We report that persistently elevated mTORC1 signaling blocks canonical D1R signaling that is dependent on DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein). The immediate downstream effector of mTORC1, ribosomal S6 kinase 1 (S6K1), phosphorylates and activates DARPP-32. Persistent elevation of mTORC1-S6K1 occludes dynamic D1R signaling downstream of DARPP-32 and blocks multiple D1R responses, including dynamic gene expression, D1R-dependent corticostriatal plasticity, and D1R behavioral responses including sociability. Candidate biomarkers of mTORC1-DARPP-32 occlusion are increased in the brain of human disease subjects in association with elevated mTORC1-S6K1, supporting a role for this mechanism in cognitive disease. CONCLUSIONS: The mTORC1-S6K1 intersection with D1R signaling provides a molecular framework to understand the effects of pathological mTORC1 activation on behavioral symptoms in neuropsychiatric disease.

Mutation-Specific and Common Phosphotyrosine Signatures of KRAS G12D and G13D Alleles.

KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as nonreceptor tyrosine kinases, MAPK kinases, and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which was found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect subtle differences between related oncogenic mutants and potentially lead to individualized treatments.

mTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation.

The microphthalmia family of transcription factors (MiT/TFEs) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via upregulated expression and activity of MiT/TFEs, whereas genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE downregulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling, respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling.

Integrated Transcriptomic and Proteomic Analysis of Human Eccrine Sweat Glands Identifies Missing and Novel Proteins.

The eccrine sweat gland is an exocrine gland that is involved in the secretion of sweat for control of temperature. Malfunction of the sweat glands can result in disorders such as miliaria, hyperhidrosis and bromhidrosis. Understanding the transcriptome and proteome of sweat glands is important for understanding their physiology and role in diseases. However, no systematic transcriptome or proteome analysis of sweat glands has yet been reported. Here, we isolated eccrine sweat glands from human skin by microdissection and performed RNA-seq and proteome analysis. In total, ∼138,000 transcripts and ∼6,100 proteins were identified. Comparison of the RNA-seq data of eccrine sweat glands to other human tissues revealed the closest resemblance to the cortex region of kidneys. The proteome data showed enrichment of proteins involved in secretion, reabsorption, and wound healing. Importantly, protein level identification of the calcium ion channel TRPV4 suggests the importance of eccrine sweat glands in re-epithelialization of wounds and prevention of dehydration. We also identified 2 previously missing proteins from our analysis. Using a proteogenomic approach, we identified 7 peptides from 5 novel genes, which we validated using synthetic peptides. Most of the novel proteins were from short open reading frames (sORFs) suggesting that many sORFs still remain to be annotated in the human genome. This study presents the first integrated analysis of the transcriptome and proteome of the human eccrine sweat gland and would become a valuable resource for studying sweat glands in physiology and disease.

Identification of novel transcripts and peptides in developing murine lens.

We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing  mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse lens including 6,990 exon skipping (ES), 2,447 alternative 3' splice site (A3SS), 1,900 alternative 5' splice site (A5SS), 1,771 mutually exclusive exons (MXE), and 173 intron retention (IR). Finally, we integrated our OMIC (Transcriptome and Proteome) datasets identifying 20 novel peptides in mouse lens. All 20 peptides were validated through matching MS/MS spectra of synthetic peptides. To the best of our knowledge, this is the first report integrating OMIC datasets to identify novel peptides in developing murine lens.

Activity-Dependent Degradation of the Nascentome by the Neuronal Membrane Proteasome.

Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase.

V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.

A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence 1417EENKVR1422 and the terminal 1478TRL1480 (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.