High resolution analysis of proteolytic substrate processing

Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, CD spectroscopy, and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other posttranslational modifications such as phosphorylation in dynamic protein complexes

Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans

VLBI celestial and terrestrial reference frames VIE2022b

Context. We present the computation of global reference frames from very long baseline interferometry (VLBI) observations at the Vienna International VLBI Service for Geodesy and Astrometry (IVS) Analysis Center (VIE) in detail. We focus on the celestial and terrestrial frames from our two latest solutions VIE2020 and VIE2022b. Aims. The current international celestial and terrestrial reference frames, ICRF3 and ITRF2020, include VLBI observations until March 2018 (at the standard geodetic and astrometric radio frequencies 2.3 and 8.4 GHz) and December 2020, respectively. We provide terrestrial and celestial reference frames including VLBI sessions until June 2022 organized by the IVS. Methods. Vienna terrestrial and celestial reference frames are computed in a common least squares adjustment of geodetic and astro-metric VLBI observations with the Vienna VLBI and Satellite Software (VieVS). Results. We provide high-precision celestial and terrestrial reference frames computed from 24 h IVS observing sessions. Our latest celestial reference frame solution VIE2022b-sx provides positions of 5407 radio sources at the frequency of 8.4 GHz. In particular, the positions of sources with few observations at the time of the ICRF3 calculation are improved. The frame also includes positions of 870 radio sources not included in ICRF3. The additional observations beyond the data used for ITRF2020 provide a more reliable estimation of positions and linear velocities of newly established VLBI Global Observing System (VGOS) telescopes