Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

The 37TrillionCells initiative for improving global healthcare via cell-based interception and precision medicine: focus on neurodegenerative diseases.

One of the main burdens in the treatment of diseases is imputable to the delay between the appearance of molecular dysfunctions in the first affected disease cells and their presence in sufficient number for detection in specific tissues or organs. This delay obviously plays in favor of disease progression to an extent that makes efficient treatments difficult, as they arrive too late. The development of a novel medical strategy, termed cell-based interception and precision medicine, seeks to identify dysfunctional cells early, when tissue damages are not apparent and symptoms not yet present, and develop therapies to treat diseases early. Central to this strategy is the use of single-cell technologies that allow detection of molecular changes in cells at the time of phenotypical bifurcation from health to disease. In this article we describe a general procedure to support such an approach applied to neurodegenerative disorders. This procedure combines four components directed towards highly complementary objectives: 1) a high-performance single-cell proteomics (SCP) method (Detect), 2) the development of disease experimental cell models and predictive computational models of cell trajectories (Understand), 3) the discovery of specific targets and personalized therapies (Cure), and 4) the creation of a community of collaborating laboratories to accelerate the development of this novel medical paradigm (Collaborate). A global initiative named 37TrillionCells (37TC) was launched to advance the development of cell-based interception and precision medicine.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex.

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.

Defining the interactomes of proteins involved in cytoskeletal dynamics using high-throughput proximity-dependent biotinylation in cellulo.

Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and a detailed protocol on how to perform BioID in mammalian cells. We also describe the mass spectrometry procedure and the bioinformatics pipeline for the data analysis. For complete details on the use and execution of this profile, please refer to Bagci et al. (2020).

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans.

Osteocalcin (OCN) is an osteoblast-derived hormone with pleiotropic physiological functions. Like many peptide hormones, OCN is subjected to post-translational modifications (PTMs) which control its activity. Here, we uncover O-glycosylation as a novel PTM present on mouse OCN and occurring on a single serine (S8) independently of its carboxylation and endoproteolysis, two other PTMs regulating this hormone. We also show that O-glycosylation increases OCN half-life in plasma ex vivo and in the circulation in vivo. Remarkably, in human OCN (hOCN), the residue corresponding to S8 is a tyrosine (Y12), which is not O-glycosylated. Yet, the Y12S mutation is sufficient to O-glycosylate hOCN and to increase its half-life in plasma compared to wildtype hOCN. These findings reveal an important species difference in OCN regulation, which may explain why serum concentrations of OCN are higher in mouse than in human.

Bones provide support and protection for organs in the body. However, over the last 15 years researchers have discovered that bones also release chemicals known as hormones, which can travel to other parts of the body and cause an effect. The cells responsible for making bone, known as osteoblasts, produce a hormone called osteocalcin which communicates with a number of different organs, including the pancreas and brain. When osteocalcin reaches the pancreas, it promotes the release of another hormone called insulin which helps regulate the levels of sugar in the blood. Osteocalcin also travels to other organs such as muscle, where it helps to degrade fats and sugars that can be converted into energy. It also has beneficial effects on the brain, and has been shown to aid memory and reduce depression. Osteocalcin has largely been studied in mice where levels are five to ten times higher than in humans. But it is unclear why this difference exists or how it alters the role of osteocalcin in humans. To answer this question, Al Rifai et al. used a range of experimental techniques to compare the structure and activity of osteocalcin in mice and humans. The experiments showed that mouse osteocalcin has a group of sugars attached to its protein structure, which prevent the hormone from being degraded by an enzyme in the blood. Human osteocalcin has a slightly different protein sequence and is therefore unable to bind to this sugar group. As a result, the osteocalcin molecules in humans are less stable and cannot last as long in the blood. Al Rifai et al. showed that when human osteocalcin was modified so the sugar group could attach, the hormone was able to stick around for much longer and reach higher levels when added to blood in the laboratory. These findings show how osteocalcin differs between human and mice. Understanding this difference is important as the effects of osteocalcin mean this hormone can be used to treat diabetes and brain disorders. Furthermore, the results reveal how the stability of osteocalcin could be improved in humans, which could potentially enhance its therapeutic effect.

DNA Binding Reorganizes the Intrinsically Disordered C-Terminal Region of PSC in Drosophila PRC1.

Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking can bring the high-affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large intrinsically disordered regions that bind nucleic acids.

Author Correction: Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.

Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.

Upstream ORF-Encoded ASDURF Is a Novel Prefoldin-like Subunit of the PAQosome.

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.

Design and Characterization of Protein E-PilA, a Candidate Fusion Antigen for Nontypeable Haemophilus influenzae Vaccine.

Nontypeable Haemophilus influenzae (NTHi) is a pathogen known for being a frequent cause of acute otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In the present study, a vaccine antigen based on the fusion of two known NTHi adhesive proteins, protein E (PE) and a pilin subunit (PilA), was developed. The quality of the combined antigen was investigated through functional, biophysical, and structural analyses. It was shown that the PE and PilA individual structures are not modified in the PE-PilA fusion and that PE-PilA assembles as a dimer in solution, reflecting PE dimerization. PE-PilA was found to bind vitronectin by enzyme-linked immunosorbent assay, as isolated PE does. Disulfide bridges were conserved and homogeneous, which was determined by peptide mapping and top-down analysis of PE, PilA, and PE-PilA molecules. Finally, the PE-PilA crystal showed a PE entity with a three-dimensional (3D) structure similar to that of the recently published isolated PE, while the structure of the PilA entity was similar to that of a 3D model elaborated from two other type 4 pilin subunits. Taken together, our observations suggest that the two tethered proteins behave independently within the chimeric molecule and display structures similar to those of the respective isolated antigens, which are important characteristics for eliciting optimal antibody-mediated immunity. PE and PilA can thus be further developed as a single fusion protein in a vaccine perspective, in the knowledge that tethering the two antigens does not perceptibly compromise the structural attributes offered by the individual antigens.

Loss of human ICOSL results in combined immunodeficiency.

Primary immunodeficiencies represent naturally occurring experimental models to decipher human immunobiology. We report a patient with combined immunodeficiency, marked by recurrent respiratory tract and DNA-based viral infections, hypogammaglobulinemia, and panlymphopenia. He also developed moderate neutropenia but without prototypical pyogenic infections. Using whole-exome sequencing, we identified a homozygous mutation in the inducible T cell costimulator ligand gene (ICOSLG; c.657C>G; p.N219K). Whereas WT ICOSL is expressed at the cell surface, the ICOSLN219K mutation abrogates surface localization: mutant protein is retained in the endoplasmic reticulum/Golgi apparatus, which is predicted to result from deleterious conformational and biochemical changes. ICOSLN219K diminished B cell costimulation of T cells, providing a compelling basis for the observed defect in antibody and memory B cell generation. Interestingly, ICOSLN219K also impaired migration of lymphocytes and neutrophils across endothelial cells, which normally express ICOSL. These defects likely contributed to the altered adaptive immunity and neutropenia observed in the patient, respectively. Our study identifies human ICOSLG deficiency as a novel cause of a combined immunodeficiency.

Posttranslational modification of proprotein convertase subtilisin/kexin type 9 is differentially regulated in response to distinct cardiometabolic treatments as revealed by targeted proteomics.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that interacts with the low-density lipoprotein (LDL) receptor at the surface of hepatocytes to regulate circulating LDL cholesterol levels. High circulating PCSK9 levels have been associated with elevated LDL cholesterol. Recently, the Food and Drug Administration of the United States approved new LDL cholesterol-lowering drugs that specifically target the inhibition of PCSK9. Similar to most human proteins, PCSK9 exists in multiple forms as it is the target of posttranslational modifications (PTMs) such as proteolytic cleavage, phosphorylation, and others, which can affect its biological activity. However, commercially available assays, such as enzyme-linked immunosorbent assays, do not discriminate between these forms. OBJECTIVE: To investigate, in 2 patient cohorts, the relationships between circulating levels of multiple forms of PCSK9 and cardiometabolic interventions or treatments known to reduce LDL cholesterol levels. METHODS: PCSK9 forms were measured in plasma: (1) in 20 patients before and 6 months after bariatric surgery and (2) in 132 patients before and 12 months after daily statin treatment. A series of specific peptides used as surrogates for various PCSK9 forms were quantified by a novel semiautomated proteomic assay termed protein affinity capture coupled to quantitative mass spectrometry. RESULTS: Bariatric surgery resulted in a decrease in the plasma level of PCSK9 prodomain (P < .05), but did not result in a significant change in other measured PCSK9 forms. Statin treatment resulted in an increase in all measured plasma PCSK9 peptides (P < .001), but a 25% decrease in the phosphorylated state of PCSK9 at S688 (P < .05). CONCLUSIONS: These unexpected findings indicate that measuring the circulating levels of the various domains and PTMs of PCSK9 provides more in depth information than total PCSK9 and that the prodomain and the phosphorylated state of S688 may represent novel biomarkers to explore in cardiometabolic diseases and response to treatment. In addition, our data generated new hypotheses on the function of PCSK9 PTMs in health and disease.

Proprotein convertase furin regulates osteocalcin and bone endocrine function.

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein.

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase.

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.