Aberrant post-translational modifications compromise human myosin motor function in old age

Abstract

Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry

“…14 Protein carbonylation is generally considered to be an irreversible PTM and a major hallmark of oxidative stress-related disorders in pathological conditions. 62 Since the human heart tissue samples used in this study are from healthy donors, it is conceivable that no carbonylation is present.…”

“…10–13 Recently, bottom-up mass spectrometry (MS) with trypsin digestion either in-gel or in-solution has been utilized for characterization of MHC. 14–17 However, the sequence coverage is very low since only a small fraction of the MHC peptides have been recovered and detected by MS. 14–17 In contrast to bottom-up MS, top-down MS analyzes intact proteins, which provides a bird’s eye view of all proteoforms 18 arising from alternative splicing of mRNAs and PTMs. 19–24 However the top-down approach still faces significant challenges for characterization of high MW proteins (> 100 kDa) mainly due to the decay of signal to noise ratio (S/N) as a function of increasing MW, 25 as well as the difficulty in protein solubilization, ionization, and fragmentation.…”

Innervation and neuromuscular control in ageing skeletal muscle

“…oxidative damage to proteins involved in cross‐bridge cycling; Li et al . ) and a shift to greater relative numbers of slow‐type MUs (Lexell & Downham, ), and to extrinsic factors namely blunted or altered activation of the fibres by the motor neuron and/or impaired junctional transmission.…”

Recent advances in mass spectrometric analysis of protein deamidation

“…Susceptibility to deamidation is also affected by protein 3D structure, local pH, temperature, ionic strength, buffer ions, and numerous other environmental variables (Robinson & Robinson, , ; Tyler‐Cross & Schirch, ). Since protein deamidation progressively disrupts structural integrity and biological activity (Lindner & Helliger, ; Lampi et al, ; Li et al, ), this modification has been strongly associated with the pathology of human aging, neurodegenerative disorders including Alzheimer's disease (AD) and cataract formation (Roher et al, ; Watanabe et al, ; Shimizu et al, ; Santa‐María et al, ; Wilmarth et al, ; Takata et al, ; Dunkelberger et al, ). However, the biological effects of protein deamidation may not be exclusively negative, since this process could also represent a mechanism of regulating biomolecule longevity and timing key host processes (Robinson & Robinson, , ).…”