Grants

NIDCR - National Institute of Dental and Craniofacial Research

ABSTRACT Osteonecrosis of the jaw (ONJ) is a damaging condition characterized by exposed and necrotic bone in the maxillofacial region, and arises most commonly in patients taking anti-resorptive medications, including bisphosphonates (BPs) and anti-RANK ligand antibodies (denosomab). This pathology, which can lead to, among other symptoms, chronic pain, jaw bone infections, and disfigurement, is most common amongst cancer patients taking high doses of these drugs, who harbor a ~5% risk of developing ONJ. Treatment strategies for ONJ vary, but none are without their downsides, making prevention of this condition, and prediction of its risk all the more important. Our goal with this proposal is to identify the mechanisms that give rise to ONJ while taking BPs, and develop a polygenic risk score based on genetic factors that we identify as associated with susceptibility to this condition. To do this, we will build upon our previous work that identified novel regulators of bisphosphonate function to investigate how use of these drugs can lead to ONJ. Using genome-wide CRISPR screens, we found over 200 genes that affect the cellular response to BPs, and identified ATRAID as encoding a lysosomal protein required for the trafficking of BPs. Loss of Atraid inhibits the effects of BPs on mouse models of osteoporosis, and in our preliminary data, variants in this gene were enriched in patients who developed ONJ. Our overall proposal applies an integrated approach to (i) elucidate mechanisms that give rise to ONJ, including which cell types, and which molecules, including ATRAID, are driving this dysregulated response, and (ii) identify genetic markers of ONJ susceptibility by conducting pharmacogenomic analyses of germline DNA collected in SWOG S0702, an NCI-funded clinical study that has available DNA and ONJ information from more than 2000 patients with metastatic bone disease treated with zoledronate, a commonly prescribed bisphosphonate, ~5% of whom developed ONJ. We will then validate these genetic loci with high-throughput cellular CRISPR screens using our previously established BP screening tools. Upon completion of this proposal, we expect to have a mechanistic understanding of ONJ and a detailed knowledge of the genetic factors that confer susceptibility to ONJ. These will have a positive impact by providing critical insights that will enable patient-centered treatment strategies for these drugs that both help alleviate concerns about ONJ among patients who need these drugs, and reduce the risk of its occurrence.

NIAID - National Institute of Allergy and Infectious Diseases

The mammalian body is continuously monitored by migratory lymphocytes that traffic via the blood to secondary lymphoid organs (SLO) in search of cognate antigens. In lymph nodes (LN) and Peyer's patches (PP), this critical surveillance mechanism depends on high endothelial venules (HEV), specialized microvessels that support multi-step adhesion cascades for circulating lymphocytes to egress into the extravascular space. HEV are found in all SLO except the spleen. In addition, HEV can arise in sites of chronic inflammation and solid tumors. These ectopic HEV mediate lymphocyte accumulation within tertiary lymphoid tissues (TLO) that may exacerbate pathologic inflammation, but also enhance tumor susceptibility to immunotherapy. Thus, pharmacologic interventions to manipulate HEV could potentially enable new drugs for cancer immunotherapy and inflammatory diseases. HEV formation in LN and PP requires the sustained action of cytokines on venular endothelial cells (EC), particularly lymphotoxin β (LTb). However, EC exposure to LTb alone is insufficient for effective HEV reprogramming, suggesting that other signals are needed. Preliminary work for this project showed a profound loss of HEV in LN and PP of mice in which EC were deficient in CD130 (gp130, IL6ST), the shared signaling subunit of a group of receptors for IL-6 family cytokines. A similar, albeit milder, phenotype was seen when EC lacked LIFR or OSMR, CD130 co-receptors that recognize lymphoma inhibitory factor (LIF) or oncostatin M (OSM), respectively. This project will explore the hypothesis that HEV formation and maintenance depends upon VEC signaling via CD130 upon ligation of LIFR and (to a lesser degree) OSMR. To test this hypothesis, Aim 1 will explore the role of endothelial CD130 in the formation and maintenance of HEV. The frequency, morphology and function of HEV in conditional 'knockout' mouse strains will be examined in steady-state SLO and in models of cancer, inflammatory bowel disease and rheumatoid arthritis. This aim will generate a comprehensive picture of the physiologic and immunological impact of intrinsic CD130 signaling in normal and pathologic HEV. Aim 2 will characterize the cytokine ligand(s) and co- receptor(s) that drive CD130 dependent HEV differentiation. Expression and localization of the known cytokines and co-receptors will be characterized in LN and PP, and HEV phenotype, function and immunological impact will be tested in conditional knockout mice lacking endothelial LIFR or OSMR. This aim will clarify the relative contribution of CD130 agonists and the LTb pathway in LN HEV formation and investigate the role of peripherally produced lymph-borne CD130 ligands vs. cytokines produced within the LN proper. Given the clinical importance of HEV formation and function for human health, this work is highly significant. If successful, the proposed studies may identify novel targets to either promote or inhibit HEV formation to treat human diseases such as cancer or chronic inflammation, respectively.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Low back pain is the world’s leading cause of disability and the degeneration of the intervertebral disc is implicated as a primary driver of pain. A hallmark of disc degeneration is loss of proteoglycans (PG), which reduces disc hydration and osmotic pressure, diminishing the ability of the disc to resist compressive loads during physical activity. This results in instability, abnormal loading conditions in adjacent joints and tissues, disc herniation and possibly low back pain. Thus, the disruption of PG homeostasis is both a key metric of disc degeneration and an opportunity for therapeutic intervention. While PG catabolism induced by inflammation and aging has been extensively studied in disc degeneration, much less is known about PG anabolism. Our global objective is to identify factors that diminish PG anabolism and lead to disc degeneration. Glucose metabolism is a key regulator of PG biosynthesis. Nucleotide sugars are essential for PG biosynthesis and are synthesized through two minor branches of glycolysis, the uronic acid pathway and the hexosamine biosynthetic pathway. ATP serves as an energy source and a building block in PG biosynthesis and is predominantly generated in disc cells by glycolysis. However, the relationship between glycolysis and disc degeneration is unclear. We hypothesize that (1) Aging decreases glycolytic enzyme activities and PG biosynthesis, while Inflammatory conditions increase glycolysis but decrease PG biosynthesis by limiting activities along the uronic acid and hexosamine pathways; (2) An age-related decrease in gluocse uptake is mediated by inflammation; (3) Disc degeneration can occur due to age and inflammation-related effects on PG biosynthesis. The hypotheses will be tested by the following specific aims. Since the imbalance of anabolism and catabolism is implicated in disc degeneration, in Aim 1, we will systematically quantify how age and inflammation impact anabolic (i.e., glucose and PG metabolism) and catabolic activities of human disc cells in vitro. Then, in Aim 2, we will investigate the in vivo correlation between glycolysis, inflammation, and disc degeneration by performing FDG PET on discs in spinal fusion patients and measuring the inflammation in discarded disc tissues. In Aim 3, we will refine our virtual human disc to include inflammation. We will then use the model to probe age and inflammation in silico and to train a lightweight AI model for image-based disc disease prediction that can integrate into the electronic health record. Combining our expertise in in vitro, in vivo, and computational analysis of disc disease will identify new therapeutic targets for disc degeneration and validate imaging and computational tools for diagnosing disc inflammation and degeneration.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

(PLEASE KEEP IN WORD, DO NOT PDF) Terminal differentiation of erythroid cells occurs in the erythroid-specific niches. The most studied erythroid niche is the erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. While studies over the past decades have identified many genes that are functionally important for EBI, the field faces major caveats. Current knowledge of EBI is predominantly derived from studies of in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. In addition, the EBI compositions in human hematopoietic tissues are unknown. In this project, we aim to uncover the anatomy, composition, and functions of EBIs in mice and humans using unbiased approaches through multiple spatial mapping technologies. Through spatial transcriptomic studies, we revealed a higher positive spatial correlation between erythroid cells and C1q+ macrophages than with other macrophages, suggesting that C1q+ macrophages are likely the EBI macrophages in mice. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed in newborn bone marrow and adult spleen under physiologic and stress conditions. We applied the same technologies to human hematopoietic tissues. In contrast to mice, we did not observe a strong positive correlation between erythroid cells and C1q+ or other macrophages in the human hematopoietic tissues. Instead, there is a strong association between erythroid progenitors and maturing erythroid cells. This erythroid self-assembled EBI structure was recapitulated in a human induced pluripotent stem cell (iPSC)-derived bone marrow organoid model. Furthermore, we identified ICAM4 as a critical erythroid surface protein that maintains erythroid-centered EBIs in humans. These preliminary studies uncover unique erythroid niches in mice and humans. Based on this evidence, we hypothesize that mouse and human EBIs have distinct structures and molecular features that help sustain terminal erythropoiesis. In this project, we propose to investigate the composition of macrophage-centered EBIs and the mechanisms of C1q in EBI macrophages in hematopoietic tissues in mice. The same approaches will be used to study ICAM4 and erythroid self-assembled EBIs in humans. Furthermore, EBI responses and their molecular mechanisms under stress and disease conditions in mice and humans will also be investigated. The success of this project will not only advance the understanding of red cell biology but also offer invaluable insights into hematopoiesis as a whole.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract The complexity of the HIV-1 transcriptome has been progressively revealed throughout recent decades using increasingly advanced RNA sequencing technologies. However, knowledge of the RNA primary sequence alone has not been sufficient to determine the importance or function of each of the over 40 highly conserved HIV-1 splice variants, which code for nine known proteins and polyproteins. RNA-protein interactions are fundamental to RNA fate and function. From transcription to cellular localization to translation of the gene product, and many steps in between, proteins interact with RNA to regulate gene expression and, in the case of HIV-1, viral replication. Notwithstanding the high significance of these splice variants in the HIV-1 life cycle, technologies for interrogating the functions, interactions, and cellular localizations of individual splice variants are woefully lacking. We propose to develop and validate a suite of powerful new tools to interrogate the functions, interactions, and cellular localizations of individual splice variants of HIV-1. For Aim 1, we will develop sensitive and multiplexed assays (HyPR-MS) to elucidate the protein interactomes of up to 20 conserved HIV-1 mRNA splice variants and advance mechanistic studies of newly identified viral RNA regulatory co-factors. In Aim 2, we will determine which HIV-1 splice variants are most critical to HIV-1 replication using virological assays and measure single- cell viral RNA abundance and subcellular localization using a multiplexed branched DNA fluorescence in situ hybridization technique (SV-FISH). These powerful new tools and strategies will be used to elucidate previously unobtainable information about HIV- 1 replication. Once developed, these same novel technologies will comprise a powerful new toolset that can be applied to splice variant investigations in other viral and cellular systems.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The HIV-1 capsid, a cone-shaped protein shell, is a promising target for antiviral interventions, with lenacapavir (LEN), a potent capsid inhibitor, demonstrating efficacy in treatment and prevention. However, resistance to LEN has been reported in both in vitro and clinical contexts. Our preliminary studies have also identified LEN-resistant mutations not previously observed. As the use of capsid inhibitors is expected to expand in the future, a deeper understanding of drug resistance—especially its evolutionary trajectories and impact on viral fitness and transmissibility—is crucial, with broad clinical implications. In this grant, we will investigate HIV-1 resistance to LEN, exploring its potential and consequences using in vitro and in vivo models. In Aim 1, we will elucidate how HIV-1 acquires increased resistance to LEN through rationally designed, accelerated in vitro evolution approaches. We will use large-scale mutant libraries combined with multiplex selection and viral adaptation to comprehensively map drug resistance pathways. These LEN- resistant variants will then be assessed in a humanized mouse model of HIV-1 LEN treatment. LEN is under investigation for pre-exposure prophylaxis; however, the transmissibility of LEN-resistant HIV-1 variants remains unclear and may impact future prevention efforts. To address this, we will evaluate the transmission efficiency of LEN-resistant variants in a humanized mouse model of mucosal transmission. CA mutations conferring LEN resistance can affect multiple CA-dependent functions, potentially influencing viral transmissibility. Correlates of transmission by LEN-resistant variants will be determined using both high- throughput phenotypic assays and in vivo experiments.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Cells within epithelial sheets undergo collective cell migration during a variety of processes including tissue morphogenesis, organ homeostasis, and cancer metastasis. However, we lack a clear understanding of how epithelial collectives sense directional cues and translate them into polarized movements, particularly in vertebrates. Through the proposed project, I will establish the zebrafish pronephros (embryonic kidney) as a tractable, in vivo model of epithelial collective cell migration. During development, the epithelial cells of the pronephric duct undergo a collective cell migration towards the anterior of the embryo, driving morphogenesis of the proximal tubule. This migration is powered by the extension and retraction of migratory protrusions at the basal cell surface. Lumenal fluid flow has been identified as the directional cue that dictates migration orientation: pronephric epithelial cells migrate against flow. Therefore, the pronephric epithelium is an ideal yet underutilized system to study how cells sense and relay directional information during collective migration, as both the upstream polarizing cue (fluid flow) and the downstream readout (anterior migration) are known. Collective migration is often regulated by polarity modules that coordinate cytoskeletal behaviors. I hypothesize that the zebrafish Fat1a/LarA/LarB proteins function as a migratory polarity module that orients pronephric protrusions in a flow-dependent manner. In Aim 1, I will test this hypothesis through live-imaging of migration in wildtype and polarity-mutant embryos. By mechanically altering pronephric flow in embryos expressing tagged polarity proteins, I will determine if the Fat1a/LarA/LarB polarity module localizes in a flow-dependent manner. Cilia have long been speculated to function as flow sensors in the pronephric epithelium. I hypothesize that mechanosensitive sensory cilia detect flow via the Pkd2/calcium signaling pathway. In Aim 2, I will test this hypothesis by performing live calcium imaging to ascertain if there is a population of cilia exhibiting flow- responsive calcium signaling. Engineering of a ciliary-trafficking Pkd2-mutant will reveal if ciliary Pkd2 is required for migration. Together, the proposed experiments will shed light on how epithelial cells translate directional cues into polarized migration, advancing our understanding of kidney development. Moreover, through completion of these aims, I will learn essential skills in zebrafish genetics, molecular cloning, and live-imaging that will be key to the success of my future independent research lab.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus responsible for Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), malignancies that predominantly affect immunocompromised individuals. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway plays a crucial role in antiviral immunity by detecting viral DNA and inducing type I interferon responses. However, KSHV has evolved mechanisms to inhibit this pathway, enabling immune evasion, viral persistence, and tumorigenesis. Despite its importance, the precise KSHV proteins that interact with and suppress cGAS-STING signaling remain largely unidentified. Traditional experimental approaches for mapping host-virus protein-protein interactions (PPIs) are labor-intensive, low-throughput, and often fail to capture the full complexity of viral immune modulation. To address this challenge, we aim to develop an AI-driven computational framework that can systematically identify KSHV-human PPIs, with a focus on viral proteins that inhibit cGAS-STING signaling. Our central hypothesis is that these interactions can be accurately predicted using an AI-based framework and experimentally validated to reveal underlying mechanisms of KSHV-driven pathogenesis. This study aims to uncover novel viral immune evasion mechanisms, enhance our understanding of KSHV pathogenesis, and uncover new therapeutic targets for KSHV-associated cancers by (1) combining deep learning and protein language models to predict high- confidence viral-host interactions using large-scale protein sequences, (2) identifying cGAS/STING-based KSHV proteins with molecular validation, and (3) characterizing functional cGAS/STING inhibitions and evaluating candidate KSHV ORFs in latency establishment or lytic replication. The to-be-developed AI models and tools will be open-sourced and widely disseminated within the community. The success of this project will have broader implications for studying immune evasion mechanisms in other pathogenic viruses, paving the way for innovative antiviral and immunotherapeutic strategies.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract This R35 proposal represents a comprehensive program that dives into the understanding of mechanosignaling, while providing training and mentorship to young scientists. The principal investigator (PI) has over a decade of experience in molecular and cellular mechanobiology in the context of both basic science and translational research. A major gap in the current field of cellular and molecular mechanobiology is that the scientific discoveries are difficult to translate to improve disease diagnosis and treatment, which is partially due to the lack of an effective approach to understand the inner works of mechanosignaling. Taking advantage of the R35 grant mechanism, this program will develop a methodology framework that can decipher principles and mechanisms of mechanosignaling at the sub-molecular level, which is achieved by combining a cutting- edge force spectroscopy platform we developed in house—the multi-fluorescence biomembrane force probe (MF-BFP)—with other in vitro and in silico approaches such as molecular and cellular engineering, super- resolution imaging, biophysical modeling, and molecular dynamics simulation. Integrins, an important family of cell mechanoreceptors, are selected as the molecular model for study due to their profound importance in general physiology and pathology. Seminal concepts under two themes will be explored. Firstly, under the theme of single receptor mechanosignaling, we will study the principles and mechanisms of how single integrins receive, transmit and transduce mechanosignals. We will identify ‘hotspots’ in the integrin structure that mediate integrin mechanosensitivity in ligand binding and conformational changes. By manipulating these hotspots via mutagenesis to directionally and finely tune the integrin mechanosensitivity, we aim to quantitatively manipulate the principles of single integrin mechanosignaling. Secondly, under the theme of receptor cis/trans-crosstalk in mechanosignaling, we will first investigate how two or more integrins cooperate to trigger mechanosignals on a single cell. Next, we will move into a new direction related to the intriguing phenomenon of ‘cell handshaking’, where we will assess the bi-directional biomechanical signal communication between two cells. Collectively, this program will provide in-depth insights into integrin-mediated mechanosignaling and inspire novel strategies for developing integrin-targeting mechanomedicines. Furthermore, the program is anticipated to establish the method and provide the know-how for deciphering mechanosignaling that are applicable to most, if not all, molecular and cellular systems. The widely application of our MF-BFP-centered methodology framework will allow us to better understand mechanobiology-related pathology in various diseases and more efficiently develop mechanobiology-inspired diagnostics and therapeutics in the future. To secure the success of the proposed research, this program will be supported by a strong network of collaborators within and outside of the PI’s home institute.

NCI - National Cancer Institute

ABSTRACT: Sgf73/ataxin-7 is a key component of an evolutionarily conserved transcriptional co-activator SAGA (Spt-Ada-Gcn5-Acetyltransferase) with two distinct enzymatic activities for chromatin modifications, and is up- and downregulated in cancer and other diseases such as eye and neurological disorders. Therefore, it is crucial to understand the regulatory networks lying upstream of Sgf73 towards understanding the pathogenesis of these diseases with targeted therapeutic interventions. However, it remains largely unknown how Sgf73 is regulated. In view of this, we carried out a set of experiments which demonstrated ubiquitylation and proteasomal degradation of Sgf73, thus revealing a novel UPS (Ubiquitin-proteasome system) regulation of Sgf73. However, the E3 ubiquitin ligase involved in such regulation of Sgf73 is yet unknown. Further, the physiological relevance of such regulation of Sgf73 on SAGA’s integrity and functions in controlling gene expression remains largely elusive. Moreover, other factors such as ubiquitin protease and conjugase involved in Sgf73 ubiquitylation and proteasomal degradation are not yet known. Answers to these important questions would fundamentally establish novel UPS regulation of Sgf73 in orchestrating gene expression, thus greatly advancing the field of gene regulation. In addition, these results would have significant impact on understanding the pathogenesis of cancer and other diseases such as eye and neurological disorders and future therapeutic development, since Sgf73 is associated with these diseases. Therefore, we propose to address above questions in this application. Specifically, we aim to (i) identify E3 ubiquitin ligase involved in Sgf73 ubiquitylation and proteasomal degradation, (ii) determine ubiquitylation site on Sgf73, (iii) determine the physiological relevance/roles of Sgf73 ubiquitylation and proteasomal degradation in regulation of SAGA’s integrity and functions in gene expression in controlling cellular health, and (iv) identify ubiquitin conjugase, ubiquitin protease and other factors involved in regulation of Sgf73 ubiquitylation and proteasomal degradation with associated functions. Collective results would identify new factors involved in regulation of Sgf73 with roles in controlling SAGA and gene expression in maintaining cellular functions, thus fundamentally advancing our understanding of gene regulation by UPS with implications in human health.

NIGMS - National Institute of General Medical Sciences

Summary My laboratory investigates how germ cells distinguish self from non-self RNAs to safeguard genome integrity across generations. Central to this work are small RNA pathways and the specialized condensates known as germ granules, which coordinate RNA regulation in the C. elegans germline. These mechanisms are crucial for protecting the genome from transposons and inappropriate gene silencing, ensuring fertility and proper development. Over the past decade, we have defined key rules that govern piRNA targeting and established how gene licensing pathways, such as CSR-1–associated small RNAs, actively protect self RNAs from piRNA-induced silencing. Our research has shown that licensing and silencing are not simply antagonistic but rely on highly orchestrated sorting decisions made within distinct sub-compartments of germ granules. We have also developed innovative tools, including synthetic piRNA systems and fluorescent reporters, that allow us to dissect small RNA activity and track regulatory outcomes in vivo. Building on these advances, our research over the next five years will address how intrinsic RNA features and associated proteins direct RNAs through specific regulatory fates—either protected or silenced. A major focus will be to understand how the spatial organization of germ granules, including functionally distinct sub-compartments, contributes to these decisions. We aim to elucidate the molecular mechanisms by which licensing pathways restrict RNAs from entering piRNA silencing domains, and how condensate interfaces regulate RNA flow and protein sorting. To achieve these goals, we are leveraging a suite of cutting-edge tools, including live- cell imaging, proximity labeling, CRISPR-based reporters, and inducible perturbation systems. These approaches will allow us to visualize dynamic RNA-protein interactions in space and time and to manipulate regulatory pathways with precision. Ultimately, our work seeks to uncover fundamental principles of small RNA-based genome defense, condensate organization, and epigenetic inheritance. These insights have broader implications for RNA regulation in other systems, and may inform the development of gene regulatory and therapeutic strategies that harness small RNAs for targeted gene regulation.

NIAID - National Institute of Allergy and Infectious Diseases

Summary The maternal-fetal interface in eutherian mammals is a uniquely complex immunological environment, tasked with the dual challenge of maintaining immune tolerance to the semi-allogenic fetus while mounting robust defenses against pathogens. Central to this protection are trophoblasts, the placental barrier cells, which constitutively secrete type III interferons (IFNLs) to restrict viral infections within the placenta and adjacent maternal tissues. Recent discoveries have highlighted that two lineage-specific placental miRNA clusters— primate-specific C19MC and rodent-specific C2MC—have independently evolved to be enriched with short interspersed nuclear elements (SINEs), such as Alu or B1 repeats. These SINE-rich regions produce double- stranded RNA (dsRNA) that triggers a viral mimicry response, activating IFNL signaling and conferring potent antiviral protection independent of classical miRNA pathways. Despite the conservation of this mechanism across species, critical gaps remain: specifically, how C19MC-derived Alu dsRNA is sensed by human trophoblasts and how this process shapes antiviral immunity at the maternal-fetal interface. To address these questions, the Totary-Jain and Coyne laboratories have joined forces to elucidate the mechanisms by which human trophoblasts constitutively produce IFNLs and defend against viral threats. Their central hypothesis posits that SINE dsRNAs derived from C19MC (in humans) and C2MC (in rodents) are recognized by distinct cellular sensors in trophoblasts, leading to the sustained release of type III IFNs. The project is organized around two specific aims. Aim 1 will define the mechanisms by which C19MC-derived Alu dsRNA is sensed and drives cell type-specific IFNL release from human trophoblasts, using advanced organoid models and genetic tools to pinpoint the relevant sensors and signaling pathways. Aim 2 will investigate the role of C19MC in placental antiviral defense and barrier integrity in vivo, leveraging innovative mouse models. Collectively, these studies aim to uncover how human C19MC orchestrates IFNL-mediated antiviral defenses, preserves placental barrier integrity, and prevents congenital viral infections, ultimately paving the way for novel strategies to bolster placental immunity.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT Aging and frailty are major contributors to chronic diseases and multimorbidity, yet the causal mechanisms driving biological aging remain poorly understood. While recent advances in multi-omics research have led to the development of biological age clocks, it is unclear which components of these models represent causal drivers of age-related diseases and which are merely correlated with aging. Furthermore, while screening tools exist that can identify existing frailty, there is a strong unmet need for screening tools that can identify patients at high future risk of frailty so that they can be identified early and offered preventative care to promote healthy aging and resilience. Using proteomic-based approaches may be a particularly successful approach, but there exist no large studies using population data to develop proteomic prediction models of future frailty. To address these gaps, I propose to bring together large-scale genetic, multi-omic, and clinical data from the UK Biobank, Multi-Ethnic Study of Atherosclerosis (MESA), FinnGen, and All of Us (combined n=70,225) to investigate the biological underpinnings of proteomic aging and develop effective clinical prediction tools for frailty risk. Specifically, this project will: (Aim 1) use genetic association data from over 500,000 individuals to identify aging proteins with causal relationships to major chronic diseases using Mendelian randomization and colocalization, (Aim 2) map the upstream epigenetic and transcriptional regulation of these proteins, and (Aim 3) develop a proteomic model to predict future risk of frailty before its onset. This work will provide significant new knowledge about the causal proteins involved in age-related multimorbidity and the upstream molecular mechanisms that regulate proteomic aging to inform possible therapeutic targets, and will develop scalable and transferable proteomics-based tools for frailty prediction to promote health and resilience in aging populations. This K99/R00 award will build upon my previous research experience in computational proteomics and population health through its emphasis on providing training in five new domains: (1) statistical genetics, (2) conducting clinically-relevant research on frailty, (3) epigenomics and transcriptomics analysis, (4) research on aging, and (5) professional development. All research will be conducted in the Analytic and Translational Genetics Unit at Massachusetts General Hospital and the Broad Institute, with mentorship from renowned scientists Drs. Mark Daly, Douglas Kiel, and Vadim Gladyshev. Additional guidance from scientific advisor Dr. Benjamin Neale will ensure exceptional guidance and support. Overall, the training environment is outstanding, the mentors and advisors are world-class, and the proposed studies address an urgent unmet need. These skills are fundamental to my goal of establishing my own lab that conducts NIH-funded research studying the genomic and proteomic basis of human aging and develops effective screening tools for use in prevention of age-related diseases and promoting healthy aging and resilience among aging populations.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The Peru Amazon region of Loreto (capital, Iquitos) accounts for 95% of all malaria in Peru, and ranks as contributing to ~25% of all malaria cases in the Americas. Peru has repeatedly launched elimination plans, most recently in 2022, based on case-finding strategies, field-deployable molecular diagnosis, and vector control measures conceived within the previously funded, NIAID-supported, Amazonian ICEMR network. Two contexts remain a challenge to achieve malaria elimination goals: (i) residual malaria transmission with sustained hypoendemicity despite the use of currently available interventions, and (ii) focal hyperendemic transmission in hard-to-reach Indigenous populations. This proposal focuses on residual urban and close-by and remote riverine transmission areas with increasing proportions of asymptomatic cases, regardless of age. Malaria transmission in these remote areas remains high and difficult to control, and the past two years have seen important rises in malaria cases. We first aim to characterize a wide range of sociodemographic, behavioral, and environmental factors that, in addition to human genetics, contribute to individual risk of malaria (re)infection and disease in the Amazon. We will estimate the relative contribution of genetic and nongenetic (potentially modifiable) factors to individual malaria risk variation and identify risk profiles in populations from residual malaria settings in Peru. Next, we will apply network analysis to analyze the interconnectedness of malaria parasite carriers between remote and more proximal sites, which we hypothesize leads to continued malaria transmission with residual malaria settings as an important source. In addition, we will track malaria transmission trends and identify hotspots in remote indigenous populations in Peru by applying field-deployable molecular diagnosis and modeling sequential antibody measurements. In summary, we will advance knowledge on individual malaria risk profiles and the impact of targeted interventions in residual malaria contexts. We will also characterize malaria transmission in hard-to-reach areas where the malaria burden is poorly understood and mitigated. Collectively, this study will make significant contributions by integrating and translating diverse data and analytic frameworks into actionable knowledge and evidence to support tailored malaria elimination efforts in Peru, elsewhere in South America, and similar contexts in the world beyond, including the protection of United States domestic and military interests.

NHLBI - National Heart Lung and Blood Institute

PROJECT ABSTRACT/SUMMARY Chronic obstructive pulmonary disease (COPD) and asthma are the two most common chronic respiratory diseases, with COPD as the third leading cause of death globally and asthma as the most common chronic disease of childhood. They are treatable, heritable, strongly affected by the environment (e.g. smoking, pollution), and exhibit some of the largest disparities in prevalence and survival among populations. Genetic and multi-omic technologies provide powerful opportunities to identify both shared and distinct heritable risk factors among asthma and COPD, connect those genetic variants with function, and delineate relationships between genetic, environment, and disease risk using multi-omic molecular assays. However, most genetic studies of lung diseases and comorbid traits to date have focused narrowly on one or few traits at a time without modeling the dense correlation between risk factors and have been vastly Eurocentric. This is problematic, as minority communities who face a higher burden of respiratory diseases disproportionately experience adverse exposures that can be challenging to study because they are often imprecisely measured, and the level of exposure that is biologically meaningful for disease onset and progression is unclear. In this R01 application, we will integrate multi-trait, multi-ancestry, and multi-omic data from massive biobanks. We will bring together genetic and phenotypic data across the All of Us Research Program, Million Veterans Program, UK Biobank, FinnGen, and other global biobanks, totaling more than 2 million participants, including approximately 240,000 COPD cases, 260,000 asthma cases, and 60,000 proteomic profiles. In Aim 1, we will conduct powerful genetic studies of lung diseases and comorbidities, model genetic correlations among related traits and ancestries to maximize genomic discovery, refine putative causal variants, and predict genetic risk accurately across populations. In Aim 2, we will connect asthma and COPD genetic risk loci to function by using their pleiotropic relationships to dissect them into sets with similar effects, then test for enrichments and colocalizations among these sets of variants with multi-omic molecular assays to elucidate their molecular mechanisms. Lastly, in Aim 3, we will bridge the gap between inherited and environmental influences to better understand phenotypic heterogeneity in asthma and COPD by leveraging proteomic and metabolomic profiles to develop scores that predict incident disease and well-known risk factors. This R01 application will deliver accurate and reproducible genetic, environmental, and multi-omic molecular predictors of asthma and COPD that will identify causal mechanisms and modifiable risk factors. Quantifying the relative importance of risk factors as proposed here is critical for advancing clinical models, nominating targeted interventions to improve public health, and ultimately reducing vast health disparities in lung disease.

NIGMS - National Institute of General Medical Sciences

Project Summary Cell survival and function depend on the ability to detect and respond to various environmental cues and intercellular signals, a process governed by complex, dynamic signaling networks. Our research seeks to understand how these networks are regulated, how they encode information through temporal and spatial dynamics, and how they drive critical cellular processes such as growth, migration, metabolism, and survival. To address these questions, we will pursue two complementary research directions. First, we will develop robust, high-throughput tools for tracking signaling network dynamics using highly multiplexed imaging of various genetically encoded fluorescent biosensors. By integrating approaches from synthetic biology, chemical biology, and computational modeling, we aim to investigate how cells interpret and integrate multiple signals to regulate growth, migration, and metabolism. We will also examine how heterogeneous signaling responses across individual cells collectively shape population-level behaviors. Second, we will explore the molecular mechanisms and functional roles of self-organized signaling activities, with a focus on the Ras-PI3K- ERK network. This network exhibits features of excitable systems, such as wave-like propagation of activity across the cell surface, and plays critical roles in proliferation, metabolism, migration, and survival. Dysregulation of this network contributes to developmental disorders, metabolic diseases, and cancer. We will examine how the distinct dynamics of PI3K isoforms relate to their specific functions, and how cell adhesion regulates self-organized signaling behavior. Together, our research takes a multidisciplinary approach to uncover fundamental principles governing signaling network dynamics, mechanisms, and functions. The experimental and computational tools developed through this work will have broad utility across the field of cellular signaling.

NHLBI - National Heart Lung and Blood Institute

Project Summary Statin therapy is widely prescribed for the management of cardiovascular diseases, yet statin-associated muscle symptoms (SAMSs) frequently lead to therapy discontinuation. Our study aims to elucidate the underlying mechanisms of SAMSs and explore potential therapeutic interventions. We hypothesize that statin-induced muscle weakness is mediated through ryanodine receptor 1 (RyR1) calcium leakage, exacerbated by the RyR1- T4706M mutation. We have access to muscle tissue and other samples from a patient who carries this RyR1- T4706M mutation and is profoundly intolerant of all statins. We further have a mouse model based on this patient’s genotype. Using cryo-electron microscopy, electrophysiological recordings, in vitro assays, and genetic mouse models, we will identify the binding sites of commonly used statins on RyR1, evaluate their effects on muscle function, and assess the potential benefits of RyR1 stabilization with ARM210 in preventing statin- induced muscle weakness. This research holds promise for identifying novel therapeutic strategies to mitigate SAMSs and improve patient tolerance of statin therapy.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT │ Chronic pain affects approximately 20% of the global population, significantly diminishing quality of life and posing a substantial socioeconomic burden. One of the most debilitating aspects of chronic pain is sleep disruption, which affects up to 90% of pain patients. Poor sleep worsens pain sensitivity and contributes to other health issues, creating a vicious cycle where pain disrupts sleep, and poor sleep exacerbates pain. Recent research has identified key brain regions involved in both pain processing and sleep regulation, particularly the centrolateral thalamus (CL) and anterior cingulate cortex (ACC), which are central to pain perception and sleep maintenance. Dysregulation of these regions in chronic pain may underlie the sleep disturbances experienced by patients. Projections from the CL to the ACC (CL→ACC) are implicated in both pain-related arousals and the affective-motivational aspects of pain. Our R21 study will be led by a collaborative team including Dr. Gregory Corder, an expert in pain biology and neural circuit mechanisms; Dr. Franz Weber, specializing in sleep electrophysiology and closed-loop optogenetics; and Dr. Raquel Adaia Sandoval Ortega, an expert in pain-sleep interactions leveraging electrophysiology, calcium imaging, and deep-learning behavior tracking. This project aims to provide the first direct evidence of how chronic pain disrupts the CL→ACC pathway, contributing to sleep fragmentation and heightened pain sensitivity. We will use advanced techniques, including in vivo calcium imaging, closed-loop optogenetics, and the LUPE deep-learning behavior tracking system, to investigate this neural circuit and assess how its modulation can alleviate both pain and sleep disturbances. Aim 1 will focus on the electrophysiological characterization of CL and ACC activity during sleep and wake states as chronic pain develops. Mice will be implanted with electroencephalography (EEG), electromyography (EMG), and intracranial electrodes targeting the CL and ACC to monitor neural oscillatory activity. Using LUPE, we will analyze pain- and sleep-related behaviors and correlate them with neural dynamics over 24-hour recording periods, mapping how nerve injury alters neural circuits over time. Aim 2 will explore bidirectional modulation of CL→ACC projection neurons using closed-loop optogenetics to either enhance or reduce pain and sleep disturbances in a peripheral nerve injury model. Excitatory and inhibitory opsins will be used to manipulate pain- active CL→ACC neurons, and we will assess the effects of optogenetic modulation on neural activity and behavior through in vivo miniscope calcium imaging of the ACC, alongside behavioral tracking with LUPE. By identifying the neural mechanisms driving pain-induced sleep disturbances, this project aims to uncover the role of thalamocortical nociceptive processes in both pain and sleep. This work will provide the foundation for future grants focused on improving sleep as a strategy to reduce chronic pain, ultimately offering new therapeutic targets and an integrated approach to pain management.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The Gasdermin (Gsdm) family consists of six paralogous genes encoding GSDMA, GSDMB, GSDMC, GSDMD, GSDME and F (GSDMF also known as PJVK or DFNB59). Gsdm proteins play a crucial role in innate immunity, particularly in inflammation and the initiation of pyroptosis, a form of programmed necrotic cell death. Unlike many other immune modulators, transcriptional regulation of Gsdm family proteins is not sufficient to execute their biological functions. Gsdms A-E share highly conserved N-terminal (N-ter) and C-terminal (C-ter) domains separated by a variable linker. The C-term exhibits self-inhibition by completely masking the N-ter hydrophobic pocket that binds lipids. Thus, even if upregulated transcriptionally, Gsdm cannot be functional until it is cleaved by a protease to release the N-ter from the self-inhibited C-term. Thus, the key for understanding Gsdm biology is the identification of the protease. This work is based on our robust unpublished data where we identified a new protease that can directly cleave both human and murine GSDMC. Rooting from this discovery, we further identified three unique features of cleaved and activated GSDMCN-ter not observed in other Gsdm family members in terms of its molecular properties, cellular functions and in vivo phenotypes. (1) In contrast to other protease-processed Gsdm family members, such as GSDM-A, -B, -D and -E, the N-terminus of GSDMC (GSDMCN-ter), processed by the newly identified protease, did NOT effectively promote pyroptosis. (2) This is because GSDMCN-ter does not localize to the plasma membrane but to other subcellular organelles. (3) GSDMCN-ter has an immune regulatory role in animal models via amplifying type-2 immune responses, unrelated to pore formation at the plasma membrane (e.g. IL-1 family member cytokine release) and pyroptosis. Accordingly, we have planned three aims to understand the mechanisms controlling these features. In Aim 1, we will analyze the non-conserved amino acids between GSDMCN-ter and other Gsdms proteins that explain the pyroptosis deficiency. We will further determine the lipid binding profile of GSDMCN-ter and try to understand how it differs from that of other Gsdms proteins. In Aim 2, we will systematically investigate the intracellular membrane structures that can be targeted by GSDMCN- ter and assess the biological consequences after GSDMCN-ter targeting. Last, in Aim 3 we will use two animal models to determine how GSDMCN-ter amplifies type 2 immune responses. In summary, Aim 1 and 2 will unveil novel mechanisms of how GSDMC functions different from other Gsdms, as well as its new roles in cell biology (not effectively promoting pyroptosis). For Aim 3, with our unique animal models, we will reveal new mechanisms of actions for GSDMC as well as therapeutic strategies to boost type 2 immunity.

NSF

This project aims to better understand how cells determine their fate when they are under stress, specifically stress in a part of the cell called the endoplasmic reticulum (ER), the cell's power plant, which produces and folds proteins. Since proteins support all cellular functions, the production of properly functioning proteins is critical to cellular health. When the ER becomes overwhelmed due to an increase in protein production needs, it triggers a response called the unfolded protein response (UPR). This type of response plays a crucial role in maintaining cell health. If the UPR goes awry, then cellular fate is altered, potentially leading to cell death. This research project develops new tools that enable the investigation of how genes behave in real-time and create systems that help cells better manage protein production under stress. Unlike older trial and-error methods, these approaches provide precise control over how cells respond, utilizing built-in feedback systems. The results of this project could lead to new ways to engineer healthier, more resilient cells. The highly multidisciplinary research environment provides broadly reaching educational and training opportunities to graduate, undergraduate, and high school students. The secretory pathway is responsible for synthesizing approximately one-third of all proteins in eukaryotic cells. As physiological demands and pathological insults constantly challenge endoplasmic reticulum (ER) homeostasis, the unfolded protein response (UPR) activates adaptive mechanisms to maintain an optimal protein production rate, reacting to diverse stimuli and leading to opposite cell fate decisions (survival or cell death). Current approaches for manipulating the UPR and investigating the link between UPR and cell fate are based on the deregulated or exogenously controlled modulation of specific UPR genes, which results in cell adaptation. This research project uses previously built feedback-regulated cells that detect the UPR status and, in response, modulate specific UPR signaling pathways to mitigate stress and enhance cell viability. This synthetic biology platform is used to investigate the relationship between the temporal progression of the UPR and cell fate. The project also engineers cells that continuously and dynamically adjust the innate cellular capacity to buffer proteotoxic stress in response to diverse stimuli, including environmental stresses (glucose deprivation, oxidative stress), the overexpression of secretory proteins, and viral particle replication and assembly. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

OD - NIH Office of the Director

Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by involvement of the skin, peripheral joints, axial skeleton, and entheses. The heterogeneity in clinical presentation and diverse tissue resident cells suggests that inflammation in different tissue compartments arises from distinct yet overlapping cellular and molecular mechanisms. However, the immune endotypes driving pathology across tissue domains remain poorly defined. Consequently, personalized treatment strategies are lacking, and despite multiple FDA-approved therapies, remission is rare, and frequent medication adjustments are needed to manage tissue-specific flares. A major barrier to targeted therapy is the absence of biomarkers that identify tissue-specific immune pathways. To address this challenge, we developed a humanized mouse model of PsA by injecting sera and PBMCs from PsA patients into immunodeficient NSG-SGM3 mice. These mice recapitulated key clinical features—arthritis, enthesitis, psoriasis, axial involvement, and dactylitis. In contrast, mice receiving samples from healthy donors did not develop disease. Spatial transcriptomics of joint tissues revealed enrichment of IL-32 and CXCL14-expressing CD8+ T cells. Strong expression of IL-17 was noted in axial tissues and dorsal root ganglia. Notably, disease development required human serum antibodies, and we identified hornerin, a defensin protein, as a candidate autoantigen. Importantly, mice humanized with blood from PsA patients unresponsive to TNF inhibitors developed skin and joint inflammation that did not respond to TNF blockade but improved with other biologics, mirroring patient treatment responses. This proposal aims to: (1) identify cellular and molecular signatures across PsA tissue domains; (2) define shared and distinct CD8+ T cell clonotypes to uncover candidate autoantigens; (3) test whether hornerin-specific autoantibodies promote inflammation via antigen presentation; (4) elucidate plasmablast-driven autoantibody production; and (5) evaluate IL-32 blockade in humanized mice derived from biologic-refractory PsA patients. These studies will provide mechanistic insights into PsA pathogenesis and enable the development of precision therapies*

NSF

The planet Earth consists of three main layers: the metallic core, the silicate mantle, and the crust. Earth’s outermost layer is a thin shell of solid rock where life flourishes. The crust under oceans, called oceanic crust, is thinner and denser, and continental crust that forms continents is thicker and lighter. The mantle lies beneath the crust and is approximately 2,900 km thick comprising 67% of the total mass of the planet and 84% of its volume. The mantle is mostly solid, but it can flow slowly over long periods of time due to high temperatures and pressures, driving plate tectonics. The Earth's innermost layer, the core, is extremely hot and dense. While the core has been a near-closed system since the time it formed, the crust and the mantle represent a very dynamic and ever-changing system constantly interacting through the continuous transfer of mass and heat. One of the most fundamental challenges in the Earth sciences is reconstruction of the chemical evolution of the crust-mantle system. Samples of some of the remaining well-preserved vestiges of the early rock record, the invaluable time capsules that may shed light on the Earth's distant geological past, have been assembled by the PI of this project over his entire scientific career; these samples will be interrogated in this project using state-of-the-art isotope and geochemical tools to help decipher the evolution of the early Earth's crust-mantle system. The work for this project will include creating a comprehensive model for the timing of formation of the continental crust and complementary chemical evolution of the mantle, significantly advancing our understanding of early Earth’s history. Therefore, it will have relevance to the long-debated question of how terrestrial planets formed and evolved and will ultimately improve our understanding of the modern Earth. This project will involve training students for their future scientific careers. This research project seeks to constrain the history of continental crustal growth and mantle depletion in the early Earth by obtaining estimates on the timing of extraction, absolute volumes, and relative proportions of continental and oceanic crust in the Archean. A novel approach of combining lithophile element isotope (Pb-Pb, 146,147Sm-142,143Nd, and 176Lu-176Hf) and trace element (e.g., rare earth elements, Nb, U, Th, Hf) abundance data obtained using the state-of-the-art analytical techniques for a global set of thirteen komatiite-basalt systems ranging in age between 3.6 and 2.7 Ga and located on five continents, will be utilized. These komatiite-basalt suites were selected because: (a) their emplacement ages have been robustly determined using several independent geochronometers, (b) a large set of high-precision short- and long-lived radiogenic isotope and lithophile trace element abundance data are available for most of them from the previous and current studies of the PI, and (c) these komatiite-basalt suites have been shown to have escaped contamination with the material of earlier-formed upper continental crust and their compositions were mostly unaffected by secondary alteration. Using the combined isotope and trace element data both available from the previous work of the PI and obtained in this study, the time-integrated Sm/Nd, Th/U, and "true" Nb/U ratios of their mantle sources will be calculated. Using these data, the absolute volumes and relative proportions of continental and oceanic crust extracted for each time frame defined by the ages of the studied komatiite-basalt systems will be estimated, and a well-constrained model of the history of continental crustal growth and complementary mantle depletion in the Archean will be generated. While working on this project, the PI will continue supporting the integration of research and education via training students, postdoctoral researchers, and visiting scientists. The PI and the undergraduates involved in this project will develop a display for the annual Maryland Day event highlighting the results from this project, presenting current ideas about early Earth history to the public and future students. This project will make a significant contribution towards building the still extremely limited high-precision radiogenic isotope and trace element database for the Earth’s earliest mafic-ultramafic rock record, and this database will be made available to the broader scientific community for the future research and development efforts. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) worldwide, yet current therapies fail to adequately address critical drivers to its progression: lipid- driven inflammation, immune dysregulation, and fibrosis that drive its progression. Triggering receptor expressed on myeloid cells 2 (TREM2), a receptor specifically expressed on macrophages, has emerged as a pivotal regulator of lipid metabolism and immune responses in metabolic disorders, but its role in DKD remains poorly understood, necessitating further investigation. Our preliminary data suggest that TREM2 exerts a protective role in DKD by mitigating lipid-driven injury and inflammation. Furthermore, we identified the TREM2 R47H mutation, a known risk factor for impaired macrophage function in neurodegenerative diseases-as a susceptibility variant for CKD. Soluble TREM2 (sTREM2), a cleavage product of TREM2, correlates with kidney function decline and inflammatory biomarkers in DKD patients, underscoring its potential as both a prognostic indicator and a therapeutic biomarker. Together, these findings establish TREM2 as a compelling target for elucidating DKD pathogenesis and for the development of innovative biomarkers and therapies. To address these knowledge gaps, we propose three synergistic aims. Aim 1 will dissect the functional and disease-modifying consequences of the TREM2 R47H mutation on macrophage function and DKD progression, leveraging genetic models and lipid-stressed macrophage assays. Next, we will define the molecular mechanisms underlying sTREM2 cleavage, its functional role in macrophage-tubule crosstalk, and its utility as a biomarker for disease progression and therapeutic response (Aim 2). Finally, we will explore the therapeutic potential of TREM2 activation, employing a novel agonist antibody to restore macrophage functionality, reduce lipid-driven injury, and improve kidney outcomes in DKD models (Aim 3). By integrating genetic, mechanistic, and therapeutic strategies, this research aims to unravel the intricate interplay between macrophage lipid metabolism and immune regulation in DKD. Moreover, it seeks to establish a robust framework for TREM2-targeted therapies and sTREM2-based biomarkers, advancing precision medicine approaches to transform the care and prognosis of patients with DKD.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY The human genome project was expected to define all protein-coding genes, but many blind spots still persist. Microproteins are a recently uncovered class of proteins composed of less than 100-150 amino acids encoded by small open reading frames (smORFs). Previous studies from our group discovered thousands of unannotated microproteins encoded on transcript regions previously assumed to be non-coding, including on annotated long non-coding RNAs and 5’- and 3’-untranslated regions (UTRs). While the majority of microproteins have yet to be functionally characterized, many have been found to regulate fundamental processes, including DNA repair, RNA metabolism, ER stress, and translation, underscoring their significance and diverse biological roles. Our long-term vision is to develop strategies for identifying functions for the remaining thousands of microproteins, which will ultimately expand our understanding of many areas of biology and potentially reveal new therapeutic approaches for different diseases. In this proposal, our goal is to leverage the unique biochemical properties of microproteins to rationally screen for which pathways and processes they participate in. First, microproteins frequently function by interacting with and regulating the activity of larger proteins and protein complexes. In addition, most microproteins are highly disordered and enriched for short linear motifs (SLiMs) and other binding motifs that would allow for interactions between their intrinsically disordered regions (IDRs) and other biomolecules. Based on decades of research on larger annotated proteins, we know that IDRs are essential to many cellular processes, including cell signaling, RNA metabolism, and subcellular organization. We also know that many microproteins harbor the same motifs as annotated proteins involved in these specific processes. We will therefore use a combination of high- throughput screens and chemical tools to identify protein interaction partners for curated sets of disordered microproteins that are likely to function in these processes. Then for each validated microprotein interaction uncovered, we will characterize the microprotein’s role in regulating the activity of the interacting protein(s) and the particular cellular process it acts on. Altogether, these studies will uncover new functional microproteins that will expand our understanding of basic biology and provide new insights into health and disease.