Diverse gene regulatory mechanisms alter rattlesnake venom gene expression at fine evolutionary scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription-factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

Dermonecrosis caused by a spitting cobra snakebite results from toxin potentiation and is prevented by the repurposed drug varespladib.

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yLuc7 protein (to mimic the lack of Luc7Ls in human U1 snRNP) and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation, and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including Thermal Proteome Profiling (TPP) and Proteome Integral Solubility Alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply TPP and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry.

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

Serine protease inhibitor, SerpinA3n regulates cardiac remodeling after myocardial infarction.

Following myocardial infarction, the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human myocardial infarction as well as non-ischemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodeling is poorly understood. We observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homolog of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix and significantly worse post infarct cardiac function. Single cell transcriptomics complemented with histology in SerpinA3n deficient animals, demonstrated increased inflammation, adverse myocyte hypertrophy and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross linking of extracellular matrix peptides consistent with increased proteolysis in SerpinA3n deficient animals. Taken together these observations demonstrate a hitherto unappreciated causal role of Serpins in regulating matrix function and post infarct cardiac remodeling.

IL7 in combination with radiotherapy stimulates a memory T-cell response to improve outcomes in HNSCC models.

Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.

Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs.

BACKGROUND: Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS: We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS: Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS: Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING: 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).

PPARα Agonism Enhances Immune Response to Radiotherapy While Dietary Oleic Acid Results in Counteraction.

PURPOSE: Head and neck cancer (HNC) improvements are stagnant, even with advances in immunotherapy. Our previous clinical trial data show that altered fatty acid (FA) metabolism correlates with outcome. We hypothesized that pharmacologic and dietary modulation of FA catabolism will affect therapeutic efficacy. EXPERIMENTAL DESIGN: We performed in vivo and in vitro experiments using PPARα agonism with fenofibrate (FF) or high oleic acid diets (OAD) with radiotherapy, generating metabolomic, proteomic, stable isotope tracing, extracellular flux analysis, and flow-cytometric data to investigate these alterations. RESULTS: FF improved antitumor efficacy of high dose per fraction radiotherapy in HNC murine models, whereas the OAD reversed this effect. FF-treated mice on the control diet had evidence of increased FA catabolism. Stable isotope tracing showed less glycolytic utilization by ex vivo CD8+ T cells. Improved efficacy correlated with intratumoral alterations in eicosanoid metabolism and downregulated mTOR and CD36. CONCLUSIONS: Metabolic intervention with increased FA catabolism improves the efficacy of HNC therapy and enhances antitumoral immune response.

Altered metabolism and DAM-signatures in female brains and microglia with aging.

Despite Alzheimer's disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo 'metabolic reprogramming', which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became 'male-like.' Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17ß-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Altered Metabolism and DAM-signatures in Female Brains and Microglia with Aging.

Despite Alzheimer's disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo 'metabolic reprogramming', which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age-and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became 'male-like.' Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17ß-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Decoding the molecular interplay in the central dogma: An overview of mass spectrometry-based methods to investigate protein-metabolite interactions.

With the emergence of next-generation nucleotide sequencing and mass spectrometry-based proteomics and metabolomics tools, we have comprehensive and scalable methods to analyze the genes, transcripts, proteins, and metabolites of a multitude of biological systems. Despite the fascinating new molecular insights at the genome, transcriptome, proteome and metabolome scale, we are still far from fully understanding cellular organization, cell cycles and biology at the molecular level. Significant advances in sensitivity and depth for both sequencing as well as mass spectrometry-based methods allow the analysis at the single cell and single molecule level. At the same time, new tools are emerging that enable the investigation of molecular interactions throughout the central dogma of molecular biology. In this review, we provide an overview of established and recently developed mass spectrometry-based tools to probe metabolite-protein interactions-from individual interaction pairs to interactions at the proteome-metabolome scale.

ECM-Focused Proteomic Analysis of Ear Punch Regeneration in Acomys Cahirinus.

In mammals, significant injury is generally followed by the formation of a fibrotic scar which provides structural integrity but fails to functionally restore damaged tissue. Spiny mice of the genus Acomys represent the first example of full skin autotomy in mammals. Acomys cahirinus has evolved extremely weak skin as a strategy to avoid predation and is able to repeatedly regenerate healthy tissue without scar after severe skin injury or full-thickness ear punches. Extracellular matrix (ECM) composition is a critical regulator of wound repair and scar formation and previous studies have suggested that alterations in its expression may be responsible for the differences in regenerative capacity observed between Mus musculus and A. cahirinus , yet analysis of this critical tissue component has been limited in previous studies by its insolubility and resistance to extraction. Here, we utilize a 2-step ECM-optimized extraction to perform proteomic analysis of tissue composition during wound repair after full-thickness ear punches in A. cahirinus and M. musculus from weeks 1 to 4 post-injury. We observe changes in a wide range of ECM proteins which have been previously implicated in wound regeneration and scar formation, including collagens, coagulation and provisional matrix proteins, and matricryptic signaling peptides. We additionally report differences in crosslinking enzyme activity and ECM protein solubility between Mus and Acomys. Furthermore, we observed rapid and sustained increases in CD206, a marker of pro-regenerative M2 macrophages, in Acomys, whereas little or no increase in CD206 was detected in Mus. Together, these findings contribute to a comprehensive understanding of tissue cues which drive the regenerative capacity of Acomys and identify a number of potential targets for future pro-regenerative therapies.

Col1a2-Deleted Mice Have Defective Type I Collagen and Secondary Reactive Cardiac Fibrosis with Altered Hypertrophic Dynamics.

RATIONALE: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. OBJECTIVE: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. METHODS AND RESULTS: We generated and characterized Col1a2-/- mice using standard gene targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFß activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2-/- mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. CONCLUSIONS: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

Native top-down proteomics reveals EGFR-ERα signaling crosstalk in breast cancer cells dissociates NUTF2 dimers to modulate ERα signaling and cell growth.

Oligomerization of proteins and their modified forms (proteoforms) produces functional protein complexes 1,2. Complexoforms are complexes that consist of the same set of proteins with different proteoforms 3. The ability to characterize these assemblies within cells is critical to understanding the molecular mechanisms involved in disease and to designing effective drugs. An outstanding biological question is how proteoforms drive function and oligomerization of complexoforms. However, tools to define endogenous proteoform-proteoform/ligand interactions are scarce 4. Here, we present a native top-down proteomics (nTDP) strategy that combines size-exclusion chromatography, nano liquid-chromatography in direct infusion mode, field asymmetric ion mobility spectrometry, and multistage mass spectrometry to identify protein assemblies (≤70 kDa) in breast cancer cells and in cells that overexpress EGFR, a resistance model of estrogen receptor-α (ER-α) targeted therapies. By identifying ~104 complexoforms from 17 protein complexes, our nTDP approach revealed several molecular features of the breast cancer proteome, including EGFR-induced dissociation of nuclear transport factor 2 (NUTF2) assemblies that modulate ER activity. Our findings show that the K4 and K55 posttranslational modification sites discovered with nTDP differentially impact the effects of NUTF2 on the inhibition of the ER signaling pathway. By characterizing endogenous proteoform-proteoform/ligand interactions, we reveal the molecular diversity of complexoforms, which allows us to propose a model for ER drug discovery in the context of designing effective inhibitors to selectively bind and disrupt the actions of targeted ER complexoforms.

Proregenerative extracellular matrix hydrogel mitigates pathological alterations of pelvic skeletal muscles after birth injury.

Pelvic floor disorders, including pelvic organ prolapse and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Childbirth, specifically vaginal delivery, has been recognized as the most important potentially modifiable risk factor for PFM injury; however, the precise mechanisms of PFM dysfunction after parturition remain elusive. In this study, we demonstrated that PFMs exhibit atrophy and fibrosis in parous women with symptomatic pelvic organ prolapse. These pathological alterations were recapitulated in a preclinical rat model of simulated birth injury (SBI). The transcriptional signature of PFMs after injury demonstrated an impairment in muscle anabolism, persistent expression of genes that promote extracellular matrix (ECM) deposition, and a sustained inflammatory response. We also evaluated the administration of acellular injectable skeletal muscle ECM hydrogel for the prevention of these pathological alterations. Treatment of PFMs with the ECM hydrogel either at the time of birth injury or 4 weeks after injury mitigated PFM atrophy and fibrosis. By evaluating gene expression, we demonstrated that these changes are mainly driven by the hydrogel-induced enhancement of endogenous myogenesis, ECM remodeling, and modulation of the immune response. This work furthers our understanding of PFM birth injury and demonstrates proof of concept for future investigations of proregenerative biomaterial approaches for the treatment of injured pelvic soft tissues.

Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis.

Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.

From volcanoes to the bench: Advantages of novel hyperthermoacidic archaeal proteases for proteomics workflows.

Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70-90 °C and pH of 2-4 with rapid digestion kinetics. The extreme HTA-Protease reaction conditions actively denature sample proteins, obviate the use of chaotropes, are largely independent of reduction and alkylation, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests alone. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights. SIGNIFICANCE: Here we introduce new capabilities for bottom-up proteomics applications with hyperthermoacidic archaeal proteases (HTA-Proteases©). HTA-Proteases have novel cleavage specificity, require no chaotropes, and allow simple one-step/five-minute sample preparations that promise to reduce variability between samples and laboratories. HTA-Proteases generate unique sets of observable peptides that are non-overlapping with tryptic peptides and significantly increase sequence coverage and available peptide targets relative to trypsin alone. HTA-Proteases show some bias for the detection and coverage of nucleic acid-binding proteins and membrane proteins relative to trypsin. These new ultra-stable enzymes function optimally in nearly boiling acidic conditions, show no autolysis, and do not require aliquoting as they are stable for years at ambient temperatures. Used independently or in conjunction with tryptic digests, HTA-Proteases offer increased proteome coverage, unique peptide targets, and brief one-step protocols amenable to automation, rapid turnaround, and high-throughput approaches.

Selective targeting of IL2Rßγ combined with radiotherapy triggers CD8- and NK-mediated immunity, abrogating metastasis in HNSCC.

The implementation of cancer immunotherapies has seen limited clinical success in head and neck squamous cell carcinoma (HNSCC). Interleukin-2 (IL-2), which modulates the survival and functionality of lymphocytes, is an attractive target for new immunotherapies but one that is limited by presence of regulatory T cells (Tregs) expressing the high-affinity IL-2Rα. The bispecific immunocytokine PD1-IL2v preferentially delivers IL-2 signaling through IL-2Rßγ on PD-1-expressing cells. Selectively targeting the intermediate-affinity IL-2Rßγ can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting the negative regulation of IL-2Rα activation on Tregs. Using radiation therapy (RT) in combination with PD1-IL2v improves local tumor control and survival, and controls metastatic spread in orthotopic HNSCC tumor models. PD1-IL2v drives systemic activation and expansion of circulating and tumor-infiltrating cytotoxic T cells and NK cells while limiting Treg-mediated immunosuppression. These data show that PD1-L2v induces durable systemic tumor control in HNSCC.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.