Expanding the Library of Covalent Cysteine Cathepsin Probes Featuring Sulfoxonium Ylide Electrophiles.

Covalent activity-based probes are invaluable tools to monitor protease activity in vitro and in vivo. We recently discovered that dimethyl sulfoxonium ylides (SYs) bind selectively to cysteine cathepsin proteases in a mechanism-dependent manner. Herein, we present the synthetic routes and characterization of an expanded library of SY probes with a greater diversity in recognition sequences. The probes exhibit a range of potency and selectivity for the cathepsin family members. We also investigated the impact of fluorophore positioning on probes bearing P1 lysine. When sulfonated cyanine 5 was attached via the lysine side chain, the resulting probe was selective for cathepsin S. When attached to the α-amine, with the side chain amine either free or Boc-protected, the probes reacted with both cathepsin S and X. Bulk in the P1 position is thus well tolerated by cathepsin S but not cathepsin X. We examined the impact of Cy5 sulfonation on probe properties, demonstrating that unsulfonated probes exhibit greater cellular uptake, which affects their relative selectivity. Finally, we demonstrated that SY probes exhibit minimal labeling of cathepsin S in freshly prepared lysates, but this increases during the prolonged incubation of lysates. This work extends our understanding of SY probes and informs future probe development.

A Qualitative Assessment of Changes in Occupational Exposures Among Healthcare Facility Workers.

Adult-onset asthma, notably prevalent among healthcare professionals, especially nurses, is often attributed to occupational factors such as exposure to cleaning agents. Studies consistently underscore the substantial role of such exposure in work-related asthma among hospital staff. We aimed to (a) identify and characterize current practices in cleaning and aerosolized medication administration; (b) assess changes in practices since a similar 2003 study of Texas healthcare workers; and (c) identify factors contributing to diverse exposures within healthcare job categories. We conducted focus groups with 38 participants in 6 healthcare settings, analyzing current practices, changes since 2003, and factors contributing to exposure diversity. We used a three-step approach for data analysis, including sociodemographic characterization, a scissor-and-sort technique for exposure description, and qualitative content analysis. Participants were primarily healthcare providers (76%) and housekeepers/cleaners (11%) who reported exposure to aerosolized medications, cleaning products, adhesives, and solvents. Participants reported transitioning from cleaning practices to new formulas with reduced odors and shifting from spray cleansers to wipes. Personal protective equipment (PPE) used during cleaning tasks varied, with training differing among job categories. Aerosolized medication administration varied among facilities, with reported medication types and protocol changes over time. The results emphasized the significance of maintaining uniform protection, disseminating knowledge, and consistently adhering to PPE protocols in the healthcare environment. Addressing the identified gaps in comprehension and potential sources of exposure variability requires additional focus on occupational health and safety initiatives.

N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain.

Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Classification of isolated versus multiple birth defects: An automated process for population-based registries.

Epidemiologic studies of birth defects often conduct separate analyses for cases that have isolated defects (e.g., spina bifida only) and cases that have multiple defects (e.g., spina bifida and a congenital heart defect). However, in some instances, cases with additional defects (e.g., spina bifida and clubfoot) may be more appropriately considered as isolated because the co-occurring defect (clubfoot) is believed to be developmentally related to the defect of interest. Determining which combinations should be considered isolated can be challenging and potentially resource intensive for registries. Thus, we developed automated classification procedures for differentiating between isolated versus multiple defects, while accounting for developmentally related defects, and applied the approach to data from the Texas Birth Defects Registry (1999-2018 deliveries). Among 235,544 nonsyndromic cases in Texas, 89% of cases were classified as having isolated defects, with proportions ranging from 25% to 92% across 43 specific defects analyzed. A large proportion of isolated cases with spina bifida (44%), lower limb reduction defects (44%), and holoprosencephaly (32%) had developmentally related defects. Overall, our findings strongly support the need to account for isolated versus multiple defects in risk factor association analyses and to account for developmentally related defects when doing so, which has implications for interpreting prior studies.

X-linked genetic associations in sporadic thoracic aortic dissection.

The male predominance in sporadic thoracic aortic aneurysm and dissection (TAD) suggests that the X chromosome contributes to TAD, but this has not been tested. We investigated whether X-linked variation-common (minor allele frequency [MAF] ≥0.01) and rare (MAF <0.01)-was associated with sporadic TAD in three cohorts of European descent (Discovery: 364 cases, 874 controls; Replication: 516 cases, 440,131 controls, and ARIC [Atherosclerosis Risk in Communities study]: 753 cases, 2247 controls). For analysis of common variants, we applied a sex-stratified logistic regression model followed by a meta-analysis of sex-specific odds ratios. Furthermore, we conducted a meta-analysis of overlapping common variants between the Discovery and Replication cohorts. For analysis of rare variants, we used a sex-stratified optimized sequence kernel association test model. Common variants results showed no statistically significant findings in the Discovery cohort. An intergenic common variant near SPANXN1 was statistically significant in the Replication cohort (p = 1.81 × 10-8). The highest signal from the meta-analysis of the Discovery and Replication cohorts was a ZNF182 intronic common variant (p = 3.5 × 10-6). In rare variants results, RTL9 reached statistical significance (p = 5.15 × 10-5). Although most of our results were statistically insignificant, our analysis is the most comprehensive X-chromosome association analysis of sporadic TAD to date.

Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Prevalence and descriptive epidemiology of choanal atresia and stenosis in Texas, 1999-2018.

Choanal atresia and stenosis are common causes of congenital nasal obstruction, but their epidemiology is poorly understood. Compared to bilateral choanal atresia/stenosis, unilateral choanal atresia/stenosis is generally diagnosed later and might be under-ascertained in birth defect registries. Data from the population-based Texas Birth Defects Registry and Texas vital records, 1999-2018, were used to assess the prevalence of choanal atresia/stenosis. Poisson regression models were used to evaluate associations with infant and maternal characteristics in two analytic groups: isolated choanal atresia/stenosis (n = 286) and isolated, bilateral choanal atresia/stenosis (n = 105). The overall prevalence of choanal atresia/stenosis was 0.92/10,000, and the prevalence of isolated choanal atresia/stenosis was 0.37/10,000 livebirths. Variables associated with choanal atresia/stenosis in one or both analytic groups included infant sex, pregnancy plurality, maternal race/ethnicity, maternal age, and maternal residence on the Texas-Mexico border. In general, adjusted prevalence ratios estimated from the two analytic groups were in the same direction but tended to be stronger in the analyses restricted to isolated, bilateral defects. Epidemiologic studies of isolated choanal atresia/stenosis should consider focusing on cases with bilateral defects, and prioritizing analyses of environmental, social, and structural factors that could account for the association with maternal residence on the Texas-Mexico border.

Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

Cleaning Tasks and Products and Asthma Among Health Care Professionals.

OBJECTIVE: Health care workers are at risk for work-related asthma, which may be affected by changes in cleaning practices. We examined associations of cleaning tasks and products with work-related asthma in health care workers in 2016, comparing them with prior results from 2003. METHODS: We estimated asthma prevalence by professional group and explored associations of self-reported asthma with job-exposure matrix-based cleaning tasks/products in a representative Texas sample of 9914 physicians, nurses, respiratory/occupational therapists, and nurse aides. RESULTS: Response rate was 34.8% (n = 2421). The weighted prevalence rates of physician-diagnosed (15.3%), work-exacerbated (4.1%), and new-onset asthma (6.7%) and bronchial hyperresponsiveness symptoms (31.1%) were similar to 2003. New-onset asthma was associated with building surface cleaning (odds ratio [OR], 1.91; 95% confidence interval [CI], 1.10-3.33), use of ortho-phthalaldehyde (OR, 1.77; 95% CI, 1.15-2.72), bleach/quaternary compounds (OR, 1.91; 95% CI, 1.10-3.33), and sprays (OR, 1.97; 95% CI, 1.12-3.47). CONCLUSION: Prevalence of asthma/bronchial hyperresponsiveness seems unchanged, whereas associations of new-onset asthma with exposures to surface cleaning remained, and decreased for instrument cleaning.

Chemical Tools to Image the Activity of PAR-Cleaving Proteases.

Protease-activated receptors (PARs) comprise a family of four G protein-coupled receptors (GPCRs) that have broad functions in health and disease. Unlike most GPCRs, PARs are uniquely activated by proteolytic cleavage of their extracellular N termini. To fully understand PAR activation and function in vivo, it is critical to also study the proteases that activate them. As proteases are heavily regulated at the post-translational level, measures of total protease abundance have limited utility. Measures of protease activity are instead required to inform their function. This review will introduce several classes of chemical probes that have been developed to measure the activation of PAR-cleaving proteases. Their strengths, weaknesses, and applications will be discussed, especially as applied to image protease activity at the whole organism, tissue, and cellular level.

Eat, prey, love: Pathogen-mediated subversion of lysosomal biology.

The mammalian lysosome is classically considered the 'garbage can' of the cell, contributing to clearance of infection through its primary function as a degradative organelle. Intracellular pathogens have evolved several strategies to evade contact with this harsh environment through subversion of endolysosomal trafficking or escape into the cytosol. Pathogens can also manipulate pathways that lead to lysosomal biogenesis or alter the abundance or activity of lysosomal content. This pathogen-driven subversion of lysosomal biology is highly dynamic and depends on a range of factors, including cell type, stage of infection, intracellular niche and pathogen load. The growing body of literature in this field highlights the nuanced and complex relationship between intracellular pathogens and the host lysosome, which is critical for our understanding of infection biology.

Protease-activated receptors in health and disease.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

Identifying syndromes in studies of structural birth defects: Guidance on classification and evaluation of potential impact.

Structural birth defects that occur in infants with syndromes may be etiologically distinct from those that occur in infants in whom there is not a recognized pattern of malformations; however, population-based registries often lack the resources to classify syndromic status via case reviews. We developed criteria to systematically identify infants with suspected syndromes, grouped by syndrome type and level of effort required for syndrome classification (e.g., text search). We applied this algorithm to the Texas Birth Defects Registry (TBDR) to describe the proportion of infants with syndromes delivered during 1999-2014. We also developed a bias analysis tool to estimate the potential percent bias resulting from including infants with syndromes in studies of risk factors. Among 207,880 cases with birth defects in the TBDR, 15% had suspected syndromes and 85% were assumed to be nonsyndromic, with a range across defect types from 28.5% (atrioventricular septal defects) to 98.9% (pyloric stenosis). Across hypothetical scenarios varying expected parameters (e.g., nonsyndromic proportion), the inclusion of syndromic cases in analyses resulted in up to 50.0% bias in prevalence ratios. In summary, we present a framework for identifying infants with syndromic conditions; implementation might harmonize syndromic classification across registries and reduce bias in association estimates.

Maternal genetic factors in the development of congenital heart defects.

Congenital heart defects (CHDs) are among the most common, serious birth defects. However, the cause of CHDs is unknown for approximately half of affected individuals and there are few prevention strategies. Although not extensively investigated, maternal genes may contribute to CHD etiology by modifying the effects of maternal exposures (e.g. medications, nutrients), contributing to maternal phenotypes that are associated with an increased risk of CHDs in offspring (e.g. diabetes), or acting as maternal effect genes. Since maternal genes could serve as a target for the primary prevention of CHDs, efforts to further define the contribution of the maternal genome to CHD etiology are warranted.

A genome-wide association study of obstructive heart defects among participants in the National Birth Defects Prevention Study.

Obstructive heart defects (OHDs) share common structural lesions in arteries and cardiac valves, accounting for ~25% of all congenital heart defects. OHDs are highly heritable, resulting from interplay among maternal exposures, genetic susceptibilities, and epigenetic phenomena. A genome-wide association study was conducted in National Birth Defects Prevention Study participants (Ndiscovery  = 3978; Nreplication  = 2507), investigating the genetic architecture of OHDs using transmission/disequilibrium tests (TDT) in complete case-parental trios (Ndiscovery_TDT  = 440; Nreplication_TDT  = 275) and case-control analyses separately in infants (Ndiscovery_CCI  = 1635; Nreplication_CCI  = 990) and mothers (case status defined by infant; Ndiscovery_CCM  = 1703; Nreplication_CCM  = 1078). In the TDT analysis, the SLC44A2 single nucleotide polymorphism (SNP) rs2360743 was significantly associated with OHD (pdiscovery  = 4.08 × 10-9 ; preplication  = 2.44 × 10-4 ). A CAPN11 SNP (rs55877192) was suggestively associated with OHD (pdiscovery  = 1.61 × 10-7 ; preplication  = 0.0016). Two other SNPs were suggestively associated (p < 1 × 10-6 ) with OHD in only the discovery sample. In the case-control analyses, no SNPs were genome-wide significant, and, even with relaxed thresholds ( × discovery < 1 × 10-5 and preplication < 0.05), only one SNP (rs188255766) in the infant analysis was associated with OHDs (pdiscovery  = 1.42 × 10-6 ; preplication  = 0.04). Additional SNPs with pdiscovery < 1 × 10-5 were in loci supporting previous findings but did not replicate. Overall, there was modest evidence of an association between rs2360743 and rs55877192 and OHD and some evidence validating previously published findings.

Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86.

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

Maternal effect genes as risk factors for congenital heart defects.

Maternal effect genes (MEGs) encode factors (e.g., RNA) in the oocyte that control embryonic development prior to activation of the embryonic genome. Over 80 mammalian MEGs have been identified, including several that have been associated with phenotypes in humans. Maternal variation in MEGs is associated with a range of adverse outcomes, which, in humans, include hydatidiform moles, zygotic cleavage failure, and offspring with multi-locus imprinting disorders. In addition, data from both animal models and humans suggest that the MEGs may be associated with structural birth defects such as congenital heart defects (CHDs). To further investigate the association between MEGs and CHDs, we conducted gene-level and gene-set analyses of known mammalian MEGs (n = 82) and two common groups of CHDs: conotruncal heart defects and left ventricular outflow tract defects. We identified 14 candidate CHD-related MEGs. These 14 MEGs include three (CDC20, KHDC3L, and TRIP13) of the 11 known human MEGs, as well as one (DNMT3A) of the eight MEGs that have been associated with structural birth defects in animal models. Our analyses add to the growing evidence that MEGs are associated with structural birth defects, in particular CHDs. Given the large proportion of individuals with structural birth defects for whom etiology of their condition is unknown, further investigations of MEGs as potential risk factors for structural birth defects are strongly warranted.

Gene-Interaction-Sensitive enrichment analysis in congenital heart disease.

BACKGROUND: Gene set enrichment analysis (GSEA) uses gene-level univariate associations to identify gene set-phenotype associations for hypothesis generation and interpretation. We propose that GSEA can be adapted to incorporate SNP and gene-level interactions. To this end, gene scores are derived by Relief-based feature importance algorithms that efficiently detect both univariate and interaction effects (MultiSURF) or exclusively interaction effects (MultiSURF*). We compare these interaction-sensitive GSEA approaches to traditional χ2 rankings in simulated genome-wide array data, and in a target and replication cohort of congenital heart disease patients with conotruncal defects (CTDs). RESULTS: In the simulation study and for both CTD datasets, both Relief-based approaches to GSEA captured more relevant and significant gene ontology terms compared to the univariate GSEA. Key terms and themes of interest include cell adhesion, migration, and signaling. A leading edge analysis highlighted semaphorins and their receptors, the Slit-Robo pathway, and other genes with roles in the secondary heart field and outflow tract development. CONCLUSIONS: Our results indicate that interaction-sensitive approaches to enrichment analysis can improve upon traditional univariate GSEA. This approach replicated univariate findings and identified additional and more robust support for the role of the secondary heart field and cardiac neural crest cell migration in the development of CTDs.