. Muthukumar Ramanathan

All Publications

• Repurposing an epithelial sodium channel inhibitor as a therapy for murine and human skin inflammation. Science translational medicine Winge, M. C., Nasrallah, M., Jackrazi, L. V., Guo, K. Q., Fuhriman, J. M., Szafran, R., Ramanathan, M., Gurevich, I., Nguyen, N. T., Siprashvili, Z., Inayathullah, M., Rajadas, J., Porter, D. F., Khavari, P. A., Butte, A. J., Marinkovich, M. P. 2024; 16 (777): eade5915

  Abstract

  Inflammatory skin disease is characterized by a pathologic interplay between skin cells and immunocytes and can result in disfiguring cutaneous lesions and systemic inflammation. Immunosuppression is commonly used to target the inflammatory component; however, these drugs are often expensive and associated with side effects. To identify previously unidentified targets, we carried out a nonbiased informatics screen to identify drug compounds with an inverse transcriptional signature to keratinocyte inflammatory signals. Using psoriasis, a prototypic inflammatory skin disease, as a model, we used pharmacologic, transcriptomic, and proteomic characterization to find that benzamil, the benzyl derivative of the US Food and Drug Administration-approved diuretic amiloride, effectively reversed keratinocyte-driven inflammatory signaling. Through three independent mouse models of skin inflammation (Rac1G12V transgenic mice, topical imiquimod, and human skin xenografts from patients with psoriasis), we found that benzamil disrupted pathogenic interactions between the small GTPase Rac1 and its adaptor NCK1. This reduced STAT3 and NF-κB signaling and downstream cytokine production in keratinocytes. Genetic knockdown of sodium channels or pharmacological inhibition by benzamil prevented excess Rac1-NCK1 binding and limited proinflammatory signaling pathway activation in patient-derived keratinocytes without systemic immunosuppression. Both systemic and topical applications of benzamil were efficacious, suggesting that it may be a potential therapeutic avenue for treating skin inflammation.

  View details for DOI 10.1126/scitranslmed.ade5915

  View details for PubMedID 39661704

• Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell Miao, W., Porter, D. F., Lopez-Pajares, V., Siprashvili, Z., Meyers, R. M., Bai, Y., Nguyen, D. T., Ko, L. A., Zarnegar, B. J., Ferguson, I. D., Mills, M. M., Jilly-Rehak, C. E., Wu, C., Yang, Y., Meyers, J. M., Hong, A. W., Reynolds, D. L., Ramanathan, M., Tao, S., Jiang, S., Flynn, R. A., Wang, Y., Nolan, G. P., Khavari, P. A. 2023; 186 (1): 80

  Abstract

  Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

  View details for DOI 10.1016/j.cell.2022.12.004

  View details for PubMedID 36608661

• PROBER identifies proteins associated with programmable sequence-specific DNA in living cells. Nature methods Mondal, S., Ramanathan, M., Miao, W., Meyers, R. M., Rao, D., Lopez-Pajares, V., Siprashvili, Z., Reynolds, D. L., Porter, D. F., Ferguson, I., Neela, P., Zhao, Y., Meservey, L. M., Guo, M., Yang, Y., Li, L., Wang, Y., Khavari, P. A. 2022; 19 (8): 959-968

  Abstract

  DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

  View details for DOI 10.1038/s41592-022-01552-w

  View details for PubMedID 35927480

• Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Yang, L. M., Costales, C., Ramanathan, M., Bulterys, P. L., Murugesan, K., Schroers-Martin, J., Alizadeh, A. A., Boyd, S. D., Brown, J. M., Nadeau, K. C., Nadimpalli, S. S., Wang, A. X., Busque, S., Pinsky, B. A., Banaei, N. 2022; 153: 105217

  Abstract

  BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response.METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naive individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination.RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P<0.001), sphingosine 1-phsophate (S1P) receptor modulators (P<0.001), mycophenolate (P=0.002), and B cell lymphoma (P=0.004); those associated with low cellular response rates included S1P receptor modulators (P<0.001) and mycophenolate (P<0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination.CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.

  View details for DOI 10.1016/j.jcv.2022.105217

  View details for PubMedID 35714462

• The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation. PLoS pathogens Meyers, J. M., Ramanathan, M., Shanderson, R. L., Beck, A., Donohue, L., Ferguson, I., Guo, M. G., Rao, D. S., Miao, W., Reynolds, D., Yang, X., Zhao, Y., Yang, Y., Blish, C., Wang, Y., Khavari, P. A. 2021; 17 (10): e1009412

  Abstract

  Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

  View details for DOI 10.1371/journal.ppat.1009412

  View details for PubMedID 34597346

• SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity. The Lancet. Infectious diseases Ramanathan, M., Ferguson, I. D., Miao, W., Khavari, P. A. 2021

  View details for DOI 10.1016/S1473-3099(21)00262-0

  View details for PubMedID 34022142

• Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site PLOS PATHOGENS Mata, M., Neben, S., Majzoub, K., Carette, J., Ramanathan, M., Khavari, P. A., Sarnow, P. 2019; 15 (5)

  View details for DOI 10.1371/journal.ppat.1007467

  View details for Web of Science ID 000471180400001

• Profiling of rotavirus 3UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication JOURNAL OF BIOLOGICAL CHEMISTRY Ren, L., Ding, S., Song, Y., Li, B., Ramanathan, M., Co, J., Amieva, M. R., Khavari, P. A., Greenberg, H. B. 2019; 294 (15): 5993–6006

  View details for DOI 10.1074/jbc.RA118.006004

  View details for Web of Science ID 000465077500030

• Methods to study RNA-protein interactions (vol 16, pg 225, 2019) NATURE METHODS Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (4): 351

  View details for DOI 10.1038/s41592-019-0366-2

  View details for Web of Science ID 000462620400034

• Methods to study RNA-protein interactions. Nature methods Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (3): 225–34

  Abstract

  Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.

  View details for PubMedID 30804549

• Profiling of rotavirus 3'UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication. The Journal of biological chemistry Ren, L., Ding, S., Song, Y., Li, B., Ramanathan, M., Co, J., Amieva, M. R., Khavari, P. A., Greenberg, H. B. 2019

  Abstract

  Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3' terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3'UTR consensus sequences. During RV infection, ATP5B bound to the RV 3'UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.

  View details for PubMedID 30770472

• RNA-protein interaction detection in living cells. Nature methods Ramanathan, M. n., Majzoub, K. n., Rao, D. S., Neela, P. H., Zarnegar, B. J., Mondal, S. n., Roth, J. G., Gai, H. n., Kovalski, J. R., Siprashvili, Z. n., Palmer, T. D., Carette, J. E., Khavari, P. A. 2018

  Abstract

  RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.

  View details for PubMedID 29400715