Proceedings of the National Academy of Sciences of the United States of America
-
Proc. Natl. Acad. Sci. U.S.A. · Aug 2018
Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.
We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. ⋯ Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.
-
Proc. Natl. Acad. Sci. U.S.A. · Aug 2018
Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.
We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. ⋯ Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.
-
Proc. Natl. Acad. Sci. U.S.A. · Jul 2018
Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH.
Endosomes have emerged as a central hub and pathogenic driver of Alzheimer's disease (AD). The earliest brain cytopathology in neurodegeneration, occurring decades before amyloid plaques and cognitive decline, is an expansion in the size and number of endosomal compartments. The strongest genetic risk factor for sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4). ⋯ HDAC inhibitors that restored NHE6 expression normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, and amyloid clearance. Thus, NHE6 is a downstream effector of ApoE4 and emerges as a promising therapeutic target in AD. These observations have prognostic implications for patients who have Christianson syndrome with loss of function mutations in NHE6 and exhibit prominent glial pathology and progressive hallmarks of neurodegeneration.
-
Proc. Natl. Acad. Sci. U.S.A. · Jul 2018
Indel-correcting DNA barcodes for high-throughput sequencing.
Many large-scale, high-throughput experiments use DNA barcodes, short DNA sequences prepended to DNA libraries, for identification of individuals in pooled biomolecule populations. However, DNA synthesis and sequencing errors confound the correct interpretation of observed barcodes and can lead to significant data loss or spurious results. Widely used error-correcting codes borrowed from computer science (e.g., Hamming, Levenshtein codes) do not properly account for insertions and deletions (indels) in DNA barcodes, even though deletions are the most common type of synthesis error. ⋯ We generate and include lists of barcodes with different lengths and error correction levels that may be useful in diverse high-throughput applications, including >106 single-error-correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with >1015 error-correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biology community.
-
Proc. Natl. Acad. Sci. U.S.A. · Jun 2018
YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.
In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. ⋯ None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.