Human alveolar macrophages display marked hypo-responsiveness to IFN-γ in both proteomic and gene expression analysis.

Alveolar macrophages (AM) perform a primary defense mechanism in the lung through phagocytosis of inhaled particles and microorganisms. AM are known to be relatively immunosuppressive consistent with the aim to limit alveolar inflammation and maintain effective gas exchange in the face of these constant challenges. How AM respond to T cell derived cytokine signals, which are critical to the defense against inhaled pathogens, is less well understood. For example, successful containment of Mycobacterium tuberculosis (Mtb) in lung macrophages is highly dependent on IFN-γ secreted by Th-1 lymphocytes, however, the proteomic IFN-γ response profile in AM remains mostly unknown. In this study, we measured IFN-γ induced protein abundance changes in human AM and autologous blood monocytes (MN). AM cells were activated by IFN-γ stimulation resulting in STAT1 phosphorylation and production of MIG/CXCL9 chemokine. However, the global proteomic response to IFN-γ in AM was dramatically limited in comparison to that of MN (9 AM vs 89 MN differentially abundant proteins). AM hypo-responsiveness was not explained by reduced JAK-STAT1 signaling nor increased SOCS1 expression. These findings suggest that AM have a tightly regulated response to IFN-γ which may prevent excessive pulmonary inflammation but may also provide a niche for the initial survival and growth of Mtb and other intracellular pathogens in the lung.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

Military Medicine and Medical Research as a Source of Inspiration and Innovation to Solve National Security and Health Challenges in the 21st Century.

The history of military medicine and research is rife with examples of novel treatments and new approaches to heal and cure soldiers and others impacted by war's devastation. In the 21st century, new threats, like climate change, are combined with traditional threats, like geopolitical conflict, to create novel challenges for our strategic interests. Extreme and inaccessible environments provide heightened risks for warfighter exposure to dangerous bacteria, viruses, and fungi, as well as exposure to toxic substances and extremes of temperature, pressure, or both providing threats to performance and eroding resilience. Back home, caring for our veterans is also a health-care priority, and the diseases of veterans increasingly overlap with the health needs of an aging society. These trends of climate change, politics, and demographics suggest performance evaluation and resilience planning and response are critical to assuring both warfighter performance and societal health. The Cleveland ecosystem, comprising several hospitals, a leading University, and one of the nation's larger Veteran's Health Administration systems, is ideal for incubating and understanding the response to these challenges. In this review, we explore the interconnections of collaborations between Defense agencies, particularly Air Force and Army and academic medical center-based investigators to drive responses to the national health security challenges facing the United States and the world.

Exploring Temporal and Sex-Linked Dysregulation in Alzheimer's Disease Phospho-Proteome.

This study aims to characterize dysregulation of phosphorylation for the 5XFAD mouse model of Alzheimer's disease (AD). Employing global phosphoproteome measurements, we analyze temporal (3, 6, 9 months) and sex-dependent effects on mouse hippocampus tissue to unveil molecular signatures associated with AD initiation and progression. Our results indicate 1.9 to 4.4 times higher phosphorylation prevalence compared to protein expression across all time points, with approximately 4.5 times greater prevalence in females compared to males at 3 and 9 months. Moreover, our findings reveal consistent phosphorylation of known AD biomarkers APOE and GFAP in 5XFAD mice, alongside novel candidates BIG3, CLCN6 and STX7, suggesting their potential as biomarkers for AD pathology. In addition, we identify PDK1 as a significantly dysregulated kinase at 9 months in females, and the regulation of gap junction activity as a key pathway associated with Alzheimer's disease across all time points. AD-Xplorer, the interactive browser of our dataset, enables exploration of AD-related changes in phosphorylation, protein expression, kinase activities, and pathways. AD-Xplorer aids in biomarker discovery and therapeutic target identification, emphasizing temporal and sex-specific nature of significant phosphoproteomic signatures. Available at: https://yilmazs.shinyapps.io/ADXplorer.

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

Temporal and Sex-Linked Protein Expression Dynamics in a Familial Model of Alzheimer's Disease.

Mouse models of Alzheimer's disease (AD) show progression through stages reflective of human pathology. Proteomics identification of temporal and sex-linked factors driving AD-related pathways can be used to dissect initiating and propagating events of AD stages to develop biomarkers or design interventions. In the present study, we conducted label-free proteome measurements of mouse hippocampus tissue with variables of time (3, 6, and 9 months), genetic background (5XFAD versus WT), and sex (equal males and females). These time points are associated with well-defined phenotypes with respect to the following: Aß42 plaque deposition, memory deficits, and neuronal loss, allowing correlation of proteome-based molecular signatures with the mouse model stages. Our data show 5XFAD mice exhibit increases in known human AD biomarkers as amyloid-beta peptide, APOE, GFAP, and ITM2B are upregulated across all time points/stages. At the same time, 23 proteins are here newly associated with Alzheimer's pathology as they are also dysregulated in 5XFAD mice. At a pathways level, the 5XFAD-specific upregulated proteins are significantly enriched for DNA damage and stress-induced senescence at 3-month only, while at 6-month, the AD-specific proteome signature is altered and significantly enriched for membrane trafficking and vesicle-mediated transport protein annotations. By 9-month, AD-specific dysregulation is also characterized by significant neuroinflammation with innate immune system, platelet activation, and hyper-reactive astrocyte-related enrichments. Aside from these temporal changes, analysis of sex-linked differences in proteome signatures uncovered novel sex and AD-associated proteins. Pathway analysis revealed sex-linked differences in the 5XFAD model to be involved in the regulation of well-known human AD-related processes of amyloid fibril formation, wound healing, lysosome biogenesis, and DNA damage. Verification of the discovery results by Western blot and parallel reaction monitoring confirm the fundamental conclusions of the study and poise the 5XFAD model for further use as a molecular tool for understanding AD.

Training the next generation of translational scientists: The Case Western Reserve University Translational Fellows Program.

Background: An important part of biomedical research is the translation of discoveries into clinical or community applications that impact patient health. For a vast majority of clinical applications and sustainable community interventions, a time-tested way to get innovations to patients is through licensing of the technology and commercial development, often through startups. While biomedical scientists and trainees are schooled in discovery research, the processes of commercialization are foreign or intimidating. Further, many trainees will not aspire to a faculty position, and other avenues of advancement are desirable. Methods: At Case Western Reserve University, we developed and launched a Translational Fellows Program to provide such training for the community, focusing specifically on graduate students and postdoctoral fellows. The goals of this program include familiarizing our trainees with the principles of entrepreneurship, product development, and startups. This is accomplished through study of their laboratory's technology to identify points of translational focus and to increase awareness to potentially move ideas and products toward societal impact. This program leverages much of our existing infrastructure and provides a mechanism for the prioritization of the translation of the technology as well as "release-time" to promote effort. Results: Launched in summer 2020, our first cohort had 3 of the 12 fellows launching startups based on their technology and submitting an National Institutes of Health Small Business Innovation Research (SBIR) proposal. At least 80% reported increased knowledge and confidence in five of six key translational competencies. Conclusion: We are now continuing and improving the program and searching for sustainable support to stabilize the program for a long-term productive future.

Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

Functional characterization of co-phosphorylation networks.

MOTIVATION: Protein phosphorylation is a ubiquitous regulatory mechanism that plays a central role in cellular signaling. According to recent estimates, up to 70% of human proteins can be phosphorylated. Therefore, the characterization of phosphorylation dynamics is critical for understanding a broad range of biological and biochemical processes. Technologies based on mass spectrometry are rapidly advancing to meet the needs for high-throughput screening of phosphorylation. These technologies enable untargeted quantification of thousands of phosphorylation sites in a given sample. Many labs are already utilizing these technologies to comprehensively characterize signaling landscapes by examining perturbations with drugs and knockdown approaches, or by assessing diverse phenotypes in cancers, neuro-degerenational diseases, infectious diseases and normal development. RESULTS: We comprehensively investigate the concept of 'co-phosphorylation' (Co-P), defined as the correlated phosphorylation of a pair of phosphosites across various biological states. We integrate nine publicly available phosphoproteomics datasets for various diseases (including breast cancer, ovarian cancer and Alzheimer's disease) and utilize functional data related to sequence, evolutionary histories, kinase annotations and pathway annotations to investigate the functional relevance of Co-P. Our results across a broad range of studies consistently show that functionally associated sites tend to exhibit significant positive or negative Co-P. Specifically, we show that Co-P can be used to predict with high precision the sites that are on the same pathway or that are targeted by the same kinase. Overall, these results establish Co-P as a useful resource for analyzing phosphoproteins in a network context, which can help extend our knowledge on cellular signaling and its dysregulation. AVAILABILITY AND IMPLEMENTATION: github.com/msayati/Cophosphorylation. This research used the publicly available datasets published by other researchers as cited in the manuscript. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optimizing biomedical discoveries as an engine of culture change in an academic medical center.

Academic discovery in biomedicine is a growing enterprise with tens of billions of dollars in research funding available to universities and hospitals. Protecting and optimizing the resultant intellectual property is required in order for the discoveries to have an impact on society. To achieve that, institutions must create a multidisciplinary, collaborative system of review and support, and utilize connections to industry partners. In this study, we outline the efforts of Case Western Reserve University, coordinated through its Clinical and Translational Science Collaborative (CTSC), to promote entrepreneurial culture, and achieve goals of product development and startup formation for biomedical and population health discoveries arising from the academic ecosystem in Cleveland. The CTSC Office of Translation and Innovation, with the university's Technology Transfer Office (TTO), helps identify and derisk promising IP while building interdisciplinary project teams to optimize the assets through key preclinical derisking steps. The benefits of coordinating funding across multiple programs, assuring dedicated project management to oversee optimizing the IP, and ensuring training to help improve proposals and encourage an entrepreneurial culture, are discussed in the context of a case study of therapeutic assets, the Council to Advance Human Health. This case study highlights best practices in academic innovation.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting.

Synchrotron X-ray footprinting (XF) is a growing structural biology technique that leverages radiation-induced chemical modifications via X-ray radiolysis of water to produce hydroxyl radicals that probe changes in macromolecular structure and dynamics in solution states of interest. The X-ray Footprinting of Biological Materials (XFP) beamline at the National Synchrotron Light Source II provides the structural biology community with access to instrumentation and expert support in the XF method, and is also a platform for development of new technological capabilities in this field. The design and implementation of a new high-throughput endstation device based around use of a 96-well PCR plate form factor and supporting diagnostic instrumentation for synchrotron XF is described. This development enables a pipeline for rapid comprehensive screening of the influence of sample chemistry on hydroxyl radical dose using a convenient fluorescent assay, illustrated here with a study of 26 organic compounds. The new high-throughput endstation device and sample evaluation pipeline now available at the XFP beamline provide the worldwide structural biology community with a robust resource for carrying out well optimized synchrotron XF studies of challenging biological systems with complex sample compositions.

Structurally distinct external solvent-exposed domains drive replication of major human prions.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques-mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.

Robust inference of kinase activity using functional networks.

Mass spectrometry enables high-throughput screening of phosphoproteins across a broad range of biological contexts. When complemented by computational algorithms, phospho-proteomic data allows the inference of kinase activity, facilitating the identification of dysregulated kinases in various diseases including cancer, Alzheimer's disease and Parkinson's disease. To enhance the reliability of kinase activity inference, we present a network-based framework, RoKAI, that integrates various sources of functional information to capture coordinated changes in signaling. Through computational experiments, we show that phosphorylation of sites in the functional neighborhood of a kinase are significantly predictive of its activity. The incorporation of this knowledge in RoKAI consistently enhances the accuracy of kinase activity inference methods while making them more robust to missing annotations and quantifications. This enables the identification of understudied kinases and will likely lead to the development of novel kinase inhibitors for targeted therapy of many diseases. RoKAI is available as web-based tool at http://rokai.io .

Co-phosphorylation networks reveal subtype-specific signaling modules in breast cancer.

MOTIVATION: Protein phosphorylation is a ubiquitous mechanism of post-translational modification that plays a central role in cellular signaling. Phosphorylation is particularly important in the context of cancer, as downregulation of tumor suppressors and upregulation of oncogenes by the dysregulation of associated kinase and phosphatase networks are shown to have key roles in tumor growth and progression. Despite recent advances that enable large-scale monitoring of protein phosphorylation, these data are not fully incorporated into such computational tasks as phenotyping and subtyping of cancers. RESULTS: We develop a network-based algorithm, CoPPNet, to enable unsupervised subtyping of cancers using phosphorylation data. For this purpose, we integrate prior knowledge on evolutionary, structural and functional association of phosphosites, kinase-substrate associations and protein-protein interactions with the correlation of phosphorylation of phosphosites across different tumor samples (a.k.a co-phosphorylation) to construct a context-specific-weighted network of phosphosites. We then mine these networks to identify subnetworks with correlated phosphorylation patterns. We apply the proposed framework to two mass-spectrometry-based phosphorylation datasets for breast cancer (BC), and observe that (i) the phosphorylation pattern of the identified subnetworks are highly correlated with clinically identified subtypes, and (ii) the identified subnetworks are highly reproducible across datasets that are derived from different studies. Our results show that integration of quantitative phosphorylation data with network frameworks can provide mechanistic insights into the differences between the signaling mechanisms that drive BC subtypes. Furthermore, the reproducibility of the identified subnetworks suggests that phosphorylation can provide robust classification of disease response and markers. AVAILABILITY AND IMPLEMENTATION: CoPPNet is available at http://compbio.case.edu/coppnet/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteins inform survival-based differences in patients with glioblastoma.

BACKGROUND: Improving the care of patients with glioblastoma (GB) requires accurate and reliable predictors of patient prognosis. Unfortunately, while protein markers are an effective readout of cellular function, proteomics has been underutilized in GB prognostic marker discovery. METHODS: For this study, GB patients were prospectively recruited and proteomics discovery using liquid chromatography-mass spectrometry analysis (LC-MS/MS) was performed for 27 patients including 13 short-term survivors (STS) (≤10 months) and 14 long-term survivors (LTS) (≥18 months). RESULTS: Proteomics discovery identified 11 941 peptides in 2495 unique proteins, with 469 proteins exhibiting significant dysregulation when comparing STS to LTS. We verified the differential abundance of 67 out of these 469 proteins in a small previously published independent dataset. Proteins involved in axon guidance were upregulated in STS compared to LTS, while those involved in p53 signaling were upregulated in LTS. We also assessed the correlation between LS MS/MS data with RNAseq data from the same discovery patients and found a low correlation between protein abundance and mRNA expression. Finally, using LC-MS/MS on a set of 18 samples from 6 patients, we quantified the intratumoral heterogeneity of more than 2256 proteins in the multisample dataset. CONCLUSIONS: These proteomic datasets and noted protein variations present a beneficial resource for better predicting patient outcome and investigating potential therapeutic targets.

Fast Protein Footprinting by X-ray Mediated Radical Trifluoromethylation.

Synchrotron radiolysis generates hydroxyl radicals (•OH) that are successful footprinting reagents. Here, we describe a new reagent for the synchrotron platform, the trifluoromethyl radical (•CF3). The radical is produced by •OH displacement of •CF3 from sodium triflinate (Langlois reagent). Upon X-ray beam exposure, the reagent labels proteins extensively without any additional chemicals on a millisecond or shorter time scale. The •CF3 is comparably reactive to •OH and produces footprinting information that complements that of •OH alone. This reagent in combination with •OH should enable novel chemistry for protein footprinting on the synchrotron platform.

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, "footprinting" studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver.

The XFP (17-BM) beamline for X-ray footprinting at NSLS-II.

Hydroxyl-radical mediated synchrotron X-ray footprinting (XF) is a powerful solution-state technique in structural biology for the study of macromolecular structure and dynamics of proteins and nucleic acids, with several synchrotron resources available to serve the XF community worldwide. The XFP (Biological X-ray Footprinting) beamline at the NSLS-II was constructed on a three-pole wiggler source at 17-BM to serve as the premier beamline for performing this technique, providing an unparalleled combination of high flux density broadband beam, flexibility in beam morphology, and sample handling capabilities specifically designed for XF experiments. The details of beamline design, beam measurements, and science commissioning results for a standard protein using the two distinct XFP endstations are presented here. XFP took first light in 2016 and is now available for general user operations through peer-reviewed proposals. Currently, beam sizes from 450 µm × 120 µm to 2.7 mm × 2.7 mm (FWHM) are available, with a flux of 1.6 × 1016 photons s-1 (measured at 325 mA ring current) in a broadband (∼5-16 keV) beam. This flux is expected to rise to 2.5 × 1016 photons s-1 at the full NSLS-II design current of 500 mA, providing an incident power density of >500 W mm-2 at full focus.

Corrigendum: Myometrial Transcriptional Signatures of Human Parturition.

[This corrects the article DOI: 10.3389/fgene.2019.00185.].