Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

Association between Brachyspira and irritable bowel syndrome with diarrhoea.

OBJECTIVE: The incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to investigate potential associations between mucus-resident microbiota and IBS symptoms. DESIGN: Mucus samples were prospectively collected from sigmoid colon biopsies from patients with IBS and healthy volunteers, and their microbial protein composition analysed by mass spectrometry. Observations were verified by immunofluorescence, electron microscopy and real-time PCR, further confirmed in a second cohort, and correlated with comprehensive profiling of clinical characteristics and mucosal immune responses. RESULTS: Metaproteomic analysis of colon mucus samples identified peptides from potentially pathogenic Brachyspira species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal Brachyspira colonisation was detected in a total of 19/62 (31%) patients with IBS from two prospective cohorts, versus 0/31 healthy volunteers (p<0.001). The prevalence of Brachyspira colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). Brachyspira attachment to the colonocyte apical membrane was observed in 20% of patients with IBS and associated with accelerated oro-anal transit, mild mucosal inflammation, mast cell activation and alterations of molecular pathways linked to bacterial uptake and ion-fluid homeostasis. Metronidazole treatment paradoxically promoted Brachyspira relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics. CONCLUSION: Mucosal Brachyspira colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for Brachyspira in the pathogenesis of IBS, particularly IBS-D.

Mucus threads from surface goblet cells clear particles from the airways.

BACKGROUND: The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. METHODS: The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. RESULTS: We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. CONCLUSIONS: These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.

The human transmembrane mucin MUC17 responds to TNFα by increased presentation at the plasma membrane.

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFα resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.

The mucus bundles responsible for airway cleaning are retained in cystic fibrosis and by cholinergic stimulation.

The beneficial effect of anticholinergic therapy for chronic lung diseases such as chronic obstructive pulmonary disease (COPD) is well documented, although cholinergic stimulation paradoxically inhibits liquid absorption, increases ciliary beat frequency and increases airway surface liquid transport.Using pig tracheobronchial explants, we quantified basal mucus transport before as well as after incubation with the clinically used antimuscarinic compound ipratropium bromide (Atrovent) and stimulation with acetylcholine.As expected, surface liquid transport was increased by acetylcholine and carbachol. In contrast, the mucus bundles secreted from the submucosal glands normally transported on the cilia were stopped from moving by acetylcholine, an effect inhibited by ipratropium bromide. Interestingly, in pigs lacking a functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel, the mucus bundles were almost immobile. As in wild-type pigs, CF surface liquid transport increased after carbachol stimulation. The stagnant CF mucus bundles were trapped on the tracheal surface attached to the surface goblet cells. Pseudomonas aeruginosa bacteria were moved by the mucus bundles in wild-type but not CF pigs.Acetylcholine thus uncouples airway surface liquid transport from transport of the surface mucus bundles as the bundles are dynamically inhibited by acetylcholine and the CFTR channel, explaining initiation of CF and COPD, and opening novel therapeutic windows.

The use of stool specimens reveals Helicobacter pylori strain diversity in a cohort of adolescents and their family members in a developed country.

Helicobacter pylori infection occurs within families but the transmission route is unknown. The use of stool specimens to genotype strains facilitates inclusion of complete families in transmission studies. Therefore, we aimed to use DNA from stools to analyze strain diversity in H. pylori infected families. We genotyped H. pylori strains using specific biprobe qPCR analysis of glmM, recA and hspA. Concentration of H. pylori organisms before DNA isolation enhanced subsequent DNA amplification. We isolated H. pylori DNA from 50 individuals in 13 families. Tm data for at least 2 of the 3 genes and sequencing of the glmM amplicon were analyzed. Similar strains were commonly found in both mothers and children and in siblings. However, 20/50 (40%) individuals had multiple strains and several individuals harbored strains not found in other family members, suggesting that even in developed countries sources of infection outside of the immediate family may exist. Whether infection occurs multiple times or one transmission event with several strains occurs is not known but future studies should aim to analyze strains from children much closer to infection onset. The presence of multiple stains in infected persons has implications for antibiotic sensitivity testing and treatment strategies.

Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.

Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.

MUC17 is an essential small intestinal glycocalyx component that is disrupted in Crohn's disease.

Crohn's disease (CD) is the chronic inflammation of the ileum and colon triggered by bacteria, but insights into molecular perturbations at the bacteria-epithelium interface are limited. We report that membrane mucin MUC17 protects small intestinal enterocytes against commensal and pathogenic bacteria. In non-inflamed CD ileum, reduced MUC17 levels correlated with a compromised glycocalyx, allowing bacterial contact with enterocytes. Muc17 deletion in mice rendered the small intestine prone to atypical infection while maintaining resistance to colitis. The loss of Muc17 resulted in spontaneous deterioration of epithelial homeostasis and extra-intestinal translocation of bacteria. Finally, Muc17-deficient mice harbored specific small intestinal bacterial taxa observed in CD. Our findings highlight MUC17 as an essential line of defense in the small intestine with relevance for early epithelial defects in CD.

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection.

The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins.

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

Understanding the Tumor Immune Microenvironment in Renal Cell Carcinoma.

Scientific understanding of how the immune microenvironment interacts with renal cell carcinoma (RCC) has substantially increased over the last decade as a result of research investigations and applying immunotherapies, which modulate how the immune system targets and eliminates RCC tumor cells. Clinically, immune checkpoint inhibitor therapy (ICI) has revolutionized the treatment of advanced clear cell RCC because of improved outcomes compared to targeted molecular therapies. From an immunologic perspective, RCC is particularly interesting because tumors are known to be highly inflamed, but the mechanisms underlying the inflammation of the tumor immune microenvironment are atypical and not well described. While technological advances in gene sequencing and cellular imaging have enabled precise characterization of RCC immune cell phenotypes, multiple theories have been suggested regarding the functional significance of immune infiltration in RCC progression. The purpose of this review is to describe the general concepts of the anti-tumor immune response and to provide a detailed summary of the current understanding of the immune response to RCC tumor development and progression. This article describes immune cell phenotypes that have been reported in the RCC microenvironment and discusses the application of RCC immunophenotyping to predict response to ICI therapy and patient survival.

Clearance of small intestinal crypts involves goblet cell mucus secretion by intracellular granule rupture and enterocyte ion transport.

Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca2+ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as "expanding secretion," efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.

Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer.

The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.

Investigating the Interaction of Helicobacter pylori with the Gastric Mucosa.

Helicobacter pylori chronically infects the gastric mucosa of humans and diseases associated with infection include gastritis, peptic ulceration, and development of gastric cancer. The organism displays a distinct tropism for the gastric mucosa of humans and for the gastric mucin MUC5AC. While the majority of organisms are found in the mucus layer overlying the epithelial cells in the stomach, adherence of the organism to the gastric epithelium is necessary for the development of disease. The interaction of H. pylori with epithelial cells results in subversion of host cell signaling and induction of an inflammatory response. Factors that influence the outcome of infection include host genetics, environmental factors, and the phenotype of the infecting strain. In this chapter, we describe cell culture assays to assess the interaction of H. pylori with epithelial cells, immunofluorescent staining to detect H. pylori in infected human gastric biopsy specimens and the use of flow cytometry to detect mucin binding to H. pylori.

IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning.

The intestine is under constant exposure to chemicals, antigens, and microorganisms from the external environment. Apical aspects of transporting epithelial cells (enterocytes) form a brush-border membrane (BBM), shaped by packed microvilli coated with a dense glycocalyx. We present evidence showing that the glycocalyx forms an epithelial barrier that prevents exogenous molecules and live bacteria from gaining access to BBM. We use a multi-omics approach to investigate the function and regulation of membrane mucins exposed on the BBM during postnatal development of the mouse small intestine. Muc17 is identified as a major membrane mucin in the glycocalyx that is specifically upregulated by IL-22 as part of an epithelial defense repertoire during weaning. High levels of IL-22 at time of weaning reprogram neonatal postmitotic progenitor enterocytes to differentiate into Muc17-expressing enterocytes, as found in the adult intestine during homeostasis. Our findings propose a role for Muc17 in epithelial barrier function in the small intestine.

ASH Research Collaborative: a real-world data infrastructure to support real-world evidence development and learning healthcare systems in hematology.

The ASH Research Collaborative is a nonprofit organization established through the American Society of Hematology's commitment to patients with hematologic conditions and the science that informs clinical care and future therapies. The ASH Research Collaborative houses 2 major initiatives: (1) the Data Hub and (2) the Clinical Trials Network (CTN). The Data Hub is a program for hematologic diseases in which networks of clinical care delivery sites are developed in specific disease areas, with individual patient data contributed through electronic health record (EHR) integration, direct data entry through electronic data capture, and external data sources. Disease-specific data models are constructed so that data can be assembled into analytic datasets and used to enhance clinical care through dashboards and other mechanisms. Initial models have been built in multiple myeloma (MM) and sickle cell disease (SCD) using the Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM) and Fast Healthcare Interoperability Resources (FHIR) standards. The Data Hub also provides a framework for development of disease-specific learning communities (LC) and testing of health care delivery strategies. The ASH Research Collaborative SCD CTN is a clinical trials accelerator that creates efficiencies in the execution of multicenter clinical trials and has been initially developed for SCD. Both components are operational, with the Data Hub actively aggregating source data and the SCD CTN reviewing study candidates. This manuscript describes processes involved in developing core features of the ASH Research Collaborative to inform the stakeholder community in preparation for expansion to additional disease areas.

Bacterial factors that mediate colonization of the stomach and virulence of Helicobacter pylori.

Helicobacter pylori is a Gram-negative microaerophilic organism that colonizes the gastric mucosa of humans. Helicobacter pylori is one of the most common infections in humans and results in the development of gastritis in all infected individuals, although the majority of people are asymptomatic. A subset of infected people develop serious disease including duodenal ulceration and gastric cancer. Helicobacter pylori exhibits many striking characteristics. It lives in the hostile environment of the stomach and displays a very strict host and tissue tropism. Despite a vigorous immune response, infection persists for the lifetime of the host unless eradicated with antimicrobials. Why H. pylori is so pathogenic in some individuals and not in others is unknown but is thought to be due to a variety of host, environmental and bacterial factors. In this review, some of the bacterial factors that mediate colonization of the gastric mucosa and play a role in the pathogenesis of this organism have been considered.

Assays to Study the Interaction of Campylobacter jejuni with the Mucosal Surface.

Mucosal colonization and overcoming the mucosal barrier are essential steps in the establishment of infection by Campylobacter jejuni. The interaction between C. jejuni and host cells, including binding and invasion, is thought to be the key virulence factor important for pathogenesis of C. jejuni infections in animals or humans. The intestinal mucosal barrier is composed of a polarized epithelium covered by a thick adherent mucus gel layer. There is a requirement for cell culture assays of infection to accurately represent the in vivo mucosal surface. In this chapter, we describe the use of a number of cell culture models and the use of polarized in vitro organ culture to examine the interaction of C. jejuni with mucosal surfaces.

Factors that mediate colonization of the human stomach by Helicobacter pylori.

Helicobacter pylori (H. pylori) colonizes the stomach of humans and causes chronic infection. The majority of bacteria live in the mucus layer overlying the gastric epithelial cells and only a small proportion of bacteria are found interacting with the epithelial cells. The bacteria living in the gastric mucus may act as a reservoir of infection for the underlying cells which is essential for the development of disease. Colonization of gastric mucus is likely to be key to the establishment of chronic infection. How H. pylori manages to colonise and survive in the hostile environment of the human stomach and avoid removal by mucus flow and killing by gastric acid is the subject of this review. We also discuss how bacterial and host factors may together go some way to explaining the susceptibility to colonization and the outcome of infection in different individuals. H. pylori infection of the gastric mucosa has become a paradigm for chronic infection. Understanding of why H. pylori is such a successful pathogen may help us understand how other bacterial species colonise mucosal surfaces and cause disease.

Copper promotes TFF1-mediated Helicobacter pylori colonization.

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.