Biology subjects

Gregorich, Z. R.

Publications and source records attributed to Gregorich, Z. R..

3 recordsLinked to original sources

bioRxiv2023

Top-down Proteomics of Myosin Light Chain Isoforms Define Chamber-Specific Expression in the Human Heart

Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an atrial and ventricular isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v, in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v and MLC-2a were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Overall, top-down proteomics allowed us an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

systems biology↗

bioRxiv2022

High Sensitivity Top-down Proteomics Captures Single Muscle Cell Heterogeneity in Large Proteoforms

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy to enable the classification of individual fiber types. This study represents the first "single-cell" top-down proteomics analysis that captures single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems. Significance StatementSingle-cell technologies are revolutionizing biology and molecular medicine by allowing direct investigation of the biological variability among individual cells. Top-down proteomics is uniquely capable of dissecting biological heterogeneity at the intact protein level. Herein, we develop a highly sensitive single-cell top-down proteomics method to reveal diverse molecular variations in large proteins (>200 kDa) among individual single muscle cells. Our results both reveal and characterize the differences in protein post-translational modifications and isoform expression possible between individual muscle cells. We further integrate functional properties with proteomics and accurately measure myosin isoforms for individual muscle fiber type classification. Our study highlights the potential of top-down proteomics for understanding how single-cell protein heterogeneity contributes to cellular functions.

biochemistry↗

bioRxiv2022

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). RBM20 is a splicing factor with two canonical domains, an RNA recognition motif (RRM) and an arginine-serine rich (RS) domain. RRM loss-of-function disrupts the splicing of RBM20 target transcripts and leads to systolic dysfunction without overt DCM, while mutations in the RS domain precipitate DCM. We show that mice lacking the RS domain (Rbm20{Delta}RS) manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized in Rbm20{Delta}RS mice but not in mice lacking the RRM, which are also deficient in RBM20 splicing. We determine that the RS domain, not other domains including the RRM, is critical for RBM20 nuclear import and define the core nuclear localization signal (NLS) within this domain. Mutation analysis of phosphorylation sites within the RS domain indicate that phosphorylation is dispensable for RBM20 nuclear import. Collectively, our findings establish disruption of the NLS in RBM20 as a causative mechanism in DCM through nucleocytoplasmic transport.

cell biology↗