Environmental allergens trigger type 2 inflammation through ripoptosome activation.

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

Control of nutrient stress-induced metabolic reprogramming by PKCζ in tumorigenesis.

Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

SOD1-Related Cerebellar Ataxia and Motor Neuron Disease: Cp Variant as Functional Modifier?

We describe a novel superoxide dismutase (SOD1) mutation-associated clinical phenotype of cerebellar ataxia and motor neuron disease with a variant in the ceruloplasmin (Cp) gene, which may have possibly contributed to a multi-factorial phenotype, supported by genetic and protein structure analyses.

Survey of Protein Sequence Embedding Models.

Derived from the natural language processing (NLP) algorithms, protein language models enable the encoding of protein sequences, which are widely diverse in length and amino acid composition, in fixed-size numerical vectors (embeddings). We surveyed representative embedding models such as Esm, Esm1b, ProtT5, and SeqVec, along with their derivatives (GoPredSim and PLAST), to conduct the following tasks in computational biology: embedding the Saccharomyces cerevisiae proteome, gene ontology (GO) annotation of the uncharacterized proteins of this organism, relating variants of human proteins to disease status, correlating mutants of beta-lactamase TEM-1 from Escherichia coli with experimentally measured antimicrobial resistance, and analyzing diverse fungal mating factors. We discuss the advances and shortcomings, differences, and concordance of the models. Of note, all of the models revealed that the uncharacterized proteins in yeast tend to be less than 200 amino acids long, contain fewer aspartates and glutamates, and are enriched for cysteine. Less than half of these proteins can be annotated with GO terms with high confidence. The distribution of the cosine similarity scores of benign and pathogenic mutations to the reference human proteins shows a statistically significant difference. The differences in embeddings of the reference TEM-1 and mutants have low to no correlation with minimal inhibitory concentrations (MIC).

Granulocyte macrophage-colony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival.

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

Bioinformatics Approaches to the Understanding of Molecular Mechanisms in Antimicrobial Resistance.

Antimicrobial resistance (AMR) is a major health concern worldwide. A better understanding of the underlying molecular mechanisms is needed. Advances in whole genome sequencing and other high-throughput unbiased instrumental technologies to study the molecular pathogenicity of infectious diseases enable the accumulation of large amounts of data that are amenable to bioinformatic analysis and the discovery of new signatures of AMR. In this work, we review representative methods published in the past five years to define major approaches developed to-date in the understanding of AMR mechanisms. Advantages and limitations for applications of these methods in clinical laboratory testing and basic research are discussed.

p62 is a key regulator of nutrient sensing in the mTORC1 pathway.

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.

Gene Expression of Pneumocystis murina after Treatment with Anidulafungin Results in Strong Signals for Sexual Reproduction, Cell Wall Integrity, and Cell Cycle Arrest, Indicating a Requirement for Ascus Formation for Proliferation.

The echinocandins are a class of antifungal agents that target ß-1,3-d-glucan (BG) biosynthesis. In the ascigerous Pneumocystis species, treatment with these drugs depletes the ascus life cycle stage, which contains BG, but large numbers of forms which do not express BG remain in the infected lungs. In the present study, the gene expression profiles of Pneumocystis murina were compared between infected, untreated mice and mice treated with anidulafungin for 2 weeks to understand the metabolism of the persisting forms. Almost 80 genes were significantly up- or downregulated. Like other fungi exposed to echinocandins, genes associated with sexual replication, cell wall integrity, cell cycle arrest, and stress comprised the strongest upregulated signals in P. murina from the treated mice. The upregulation of the P. murina ß-1,3-d-glucan endohydrolase and endo-1,3-glucanase was notable and may explain the disappearance of the existing asci in the lungs of treated mice since both enzymes can degrade BG. The biochemical measurement of BG in the lungs of treated mice and fluorescence microscopy with an anti-BG antibody supported the loss of BG. Downregulated signals included genes involved in cell replication, genome stability, and ribosomal biogenesis and function and the Pneumocystis-specific genes encoding the major surface glycoproteins (Msg). These studies suggest that P. murina attempted to undergo sexual replication in response to a stressed environment and was halted in any type of proliferative cycle, likely due to a lack of BG. Asci appear to be a required part of the life cycle stage of Pneumocystis, and BG may be needed to facilitate progression through the life cycle via sexual replication.

Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA.

Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle, which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids, producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGAG/CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.

Congenital heart disease and aortic arch variants associated with mutation in PHOX2B.

PURPOSE: Congenital central hypoventilation syndrome (CCHS, OMIM 209880) is a rare autosomal dominant disorder caused by mutation in PHOX2B that manifests as a consequence of abnormal neural crest cell migration during embryogenesis. Unlike other neurocristopathies, however, its impact on the cardiovascular system has not been previously assessed. This study was an effort to characterize the association between congenital heart disease (CHD) and mutations in PHOX2B in patients with CCHS. METHODS: A retrospective review of patients with CCHS in conjunction with functional analysis of PHOX2B mutations associated with CHD was performed. To substantiate functional implications of identified variants, we conducted protein structure analyses and in silico mutagenesis were conducted. RESULTS: The prevalence of CHD among patients with CCHS was significantly greater (30%; p < 0.001) than that of the current estimated prevalence of CHD. The majority of patients had anomalies involving the proximal aortic arch and/or proximal coronary arteries. Variants associated with CHD in this cohort appear to disrupt DNA binding of PHOX2B via alteration of its homeobox domain. CONCLUSION: This is the first report of an association between CHD and mutation in PHOX2B. Results are highly suggestive that alteration or elimination of the homeobox domain conveys significant risk for associated CHD or aortic arch variation.

The 14th International Workshops on Opportunistic Protists (IWOP 14).

The 14th International Workshops on Opportunistic Protists (IWOP-14) was held August 10-12, 2017 in Cincinnati, OH, USA. The IWOP meetings focus on opportunistic protists (OIs); for example, free-living amoebae, Pneumocystis spp., Cryptosporidium spp., Toxoplasma, the Microsporidia, and kinetoplastid flagellates. The highlights of Pneumocystis spp. research included the reports of primary homothallism for mating; a potential requirement for sexual replication in its life cycle; a new antigen on the surface of small asci; roles for CLRs, Dectin-1, and Mincle in host responses; and identification of MSG families and mechanisms used for surface variation. Studies of Cryptosporidia spp. included comparative genomics, a new cryopreservation method; the role of mucin in attachment and invasion, and epidemiological surveys illustrating species diversity in animals. One of the five identified proteins in the polar tube of Microsporidia, PTP4, was shown to play a role in host infection. Zebrafish were used as a low cost vertebrate animal model for an evaluation of potential anti-toxoplasma drugs. Folk medicine compounds with anti-toxoplasma activity were presented, and reports on the chronic toxoplasma infection provided evidence for increased tractability for the study of this difficult life cycle stage. Escape from the parasitophorus vacuole and cell cycle regulation were the topics of the study in the acute phase.

Calpain-14 and its association with eosinophilic esophagitis.

Calpains are a family of intracellular, calcium-dependent cysteine proteases involved in a variety of regulatory processes, including cytoskeletal dynamics, cell-cycle progression, signal transduction, gene expression, and apoptosis. These enzymes have been implicated in a number of disease processes, notably for this review involving eosinophilic tissue inflammation, such as eosinophilic esophagitis (EoE), a chronic inflammatory disorder triggered by allergic hypersensitivity to food and associated with genetic variants in calpain 14 (CAPN14). Herein we review the genetic, structural, and biochemical properties of CAPN14 and its gene product CAPN14, and its emerging role in patients with EoE. The CAPN14 gene is localized at chromosome 2p23.1-p21 and is most homologous to CAPN13 (36% sequence identity), which is located 365 kb downstream of CAPN14. Structurally, CAPN14 has classical calpain motifs, including a cysteine protease core. In comparison with other human calpains, CAPN14 has a unique expression pattern, with the highest levels in the upper gastrointestinal tract, particularly in the squamous epithelium of the esophagus. The CAPN14 gene is positioned in an epigenetic hotspot regulated by IL-13, a TH2 cytokine with increased levels in patients with EoE that has been shown to be a mediator of the disease. CAPN14 induces disruptive effects on the esophageal epithelium by impairing epithelial barrier function in association with loss of desmoglein-1 expression and has a regulatory role in repairing epithelial changes induced by IL-13. Thus CAPN14 is a unique protease with distinct tissue-specific expression and function in patients with EoE and is a potential therapeutic target for EoE and related eosinophilic and allergic diseases.

The Epstein-Barr Virus B-ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5-Methylcytosine and 5-Hydroxymethylcytosine.

The Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the sequence specific DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1) DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine (5mC) in one strand and cytosine in the second strand, (3) DNA with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand, and (4) DNA in which both cytosines in all CG dinucleotides contain 5mC. We compared these data to PBM data for three additional B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M = 5mC), were preferentially bound. 5mC inhibits binding of Zta to both TRE motif half-sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer more strongly bind the C/EBP half-site tetranucleotide GCAA when it contains 5mC. Zta also binds dsDNA sequences containing 5hmC in one strand, although the effect is less dramatic than that observed for 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, uncovering the potential for discovery of new viral and host regulatory programs controlled by EBV.

CoeViz: a web-based tool for coevolution analysis of protein residues.

BACKGROUND: Proteins generally perform their function in a folded state. Residues forming an active site, whether it is a catalytic center or interaction interface, are frequently distant in a protein sequence. Hence, traditional sequence-based prediction methods focusing on a single residue (or a short window of residues) at a time may have difficulties in identifying and clustering the residues constituting a functional site, especially when a protein has multiple functions. Evolutionary information encoded in multiple sequence alignments is known to greatly improve sequence-based predictions. Identification of coevolving residues further advances the protein structure and function annotation by revealing cooperative pairs and higher order groupings of residues. RESULTS: We present a new web-based tool (CoeViz) that provides a versatile analysis and visualization of pairwise coevolution of amino acid residues. The tool computes three covariance metrics: mutual information, chi-square statistic, Pearson correlation, and one conservation metric: joint Shannon entropy. Implemented adjustments of covariance scores include phylogeny correction, corrections for sequence dissimilarity and alignment gaps, and the average product correction. Visualization of residue relationships is enhanced by hierarchical cluster trees, heat maps, circular diagrams, and the residue highlighting in protein sequence and 3D structure. Unlike other existing tools, CoeViz is not limited to analyzing conserved domains or protein families and can process long, unstructured and multi-domain proteins thousands of residues long. Two examples are provided to illustrate the use of the tool for identification of residues (1) involved in enzymatic function, (2) forming short linear functional motifs, and (3) constituting a structural domain. CONCLUSIONS: CoeViz represents a practical resource for a quick sequence-based protein annotation for molecular biologists, e.g., for identifying putative functional clusters of residues and structural domains. CoeViz also can serve computational biologists as a resource of coevolution matrices, e.g., for developing machine learning-based prediction models. The presented tool is integrated in the POLYVIEW-2D server (http://polyview.cchmc.org/) and available from resulting pages of POLYVIEW-2D.

Pneumocystis murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30-60%mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed Neutrophil Extracellular Trap (NET) presence in the lungs of the P. murina -infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina . While isolated NETs did not compromise P. murina viability, our data highlight the potential role of neutrophils in promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.

Pneumocystis murina promotes inflammasome formation and NETosis during Pneumocystis pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. Isolated NETs compromised P. murina viability in vitro, highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

Insights into copper sensing and tolerance in Pneumocystis species.

Introduction: Pneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance. Methods and results: The query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 µM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies - homology-based annotation, SEC-ICP-MS/IMAC/LC-MS/MS, and RNA-seq - yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods. Discussion: The insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.

Anti-CELA1 antibody KF4 prevents emphysema by inhibiting stretch-mediated remodeling.

There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non-AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1-/- mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix-stabilizing therapy in emphysema.

Tumor-suppressing function of caspase-2 requires catalytic site Cys-320 and site Ser-139 in mice.

The multifunctional caspase-2 protein is involved in apoptosis, NF-κB regulation, and tumor suppression in mice. However, the mechanisms of caspase-2 responsible for tumor suppression remain unclear. Here we identified two sites of caspase-2, the catalytic Cys-320 site and the Ser-139 site, to be important for suppression of cellular transformation and tumorigenesis. Using SV40- and K-Ras-transformed caspase-2 KO mouse embryonic fibroblast cells reconstituted with expression of wild-type, catalytic dead (C320A), or Ser-139 (S139A) mutant caspase-2, we demonstrated that similar to caspase-2 deficiency, when Cys-320 and Ser-139 were mutated, caspase-2 lost its ability to inhibit cellular transformation and tumorigenesis. These mutant cells exhibited enhanced cell proliferation, elevated clonogenic activity, accelerated anchorage-independent growth, and transformation and were highly tumorigenic, rapidly producing large tumors in athymic nude mice. Investigation into the underlying mechanism showed that these two residues are needed for caspase-2 to suppress NF-κB activity, promote apoptosis, and sustain the G(2)/M checkpoint following DNA damage induction. In addition, tumors in nude mice derived from the two mutant cell lines had higher constitutive NF-κB activity and elevated expression of NF-κB targets of antiapoptotic proteins Bcl-xL, XIAP, and cIAP2. A reduction in caspase-2 mRNA was associated with multiple types of cancers in patients. Together, these observations suggest the combined functions of caspase-2 in suppressing NF-κB activation, promoting apoptosis, and sustaining G(2)/M checkpoint contribute to caspase-2 tumor-suppressing function and that caspase-2 may also impact tumor suppression in humans. These findings provide insight into tumor suppression at the cross-roads of apoptosis, cell cycle checkpoint, and NF-κB pathways.