More Like this

1. Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency

   https://doi.org/10.1161/CIRCRESAHA.120.316966  

   Ward, Tarsha ; Tai, Warren ; Morton, Sarah ; Impens, Francis ; Van Damme, Petra ; Van Haver, Delphi ; Timmerman, Evy ; Venturini, Gabriela ; Zhang, Kehan ; Jang, Min Young ; et al ( April 2021 , Circulation Research)

   Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function,more »compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.« less
2. Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin

   https://doi.org/10.1126/scitranslmed.aat1199  

   Toepfer, Christopher N. ; Wakimoto, Hiroko ; Garfinkel, Amanda C. ; McDonough, Barbara ; Liao, Dan ; Jiang, Jianming ; Tai, Angela C. ; Gorham, Joshua M. ; Lunde, Ida G. ; Lun, Mingyue ; et al ( January 2019 , Science Translational Medicine)

   The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis andmore »thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.« less
3. In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

   https://doi.org/10.1128/JVI.01049-19  

   Steger, Courtney L. ; Brown, Mackenzie L. ; Sullivan, Owen M. ; Boudreaux, Crystal E. ; Cohen, Courtney A. ; LaConte, Leslie E. ; McDonald, Sarah M. ( October 2019 , Journal of Virology)

   López, Susana (Ed.)

   ABSTRACT The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-foldmore »axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation. IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.« less
4. Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast

   https://doi.org/10.1126/scisignal.aaz4401  

   Ghosh, Chandrima ; Uppala, Jagadeesh Kumar ; Sathe, Leena ; Hammond, Charlotte I. ; Anshu, Ashish ; Pokkuluri, P. Raj ; Turk, Benjamin E. ; Dey, Madhusudan ( May 2021 , Science Signaling)

   During cellular stress in the budding yeastSaccharomyces cerevisiae, an endoplasmic reticulum (ER)–resident dual kinase and RNase Ire1 splices an intron fromHAC1mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute toHAC1mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediatedHAC1mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3′ untranslated region (3′UTR) ofHAC1mRNA, and a yeast strain lacking bothmore »Pal1 and Pal2 was deficient inHAC1mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in apal1Δpal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinatesHAC1mRNA processing and ER homeostasis.

   « less
5. TrmB family transcription factor as a thiol-based regulator of oxidative stress response

   https://doi.org/10.1101/2022.03.05.483106  

   Mondragon, P. Hwang ( January 2022 , bioRxiv)

   Oxidative stress causes cellular damage including DNA mutations, protein dysfunction and loss of membrane integrity. Here we discovered TrmB (transcription regulator of mal operon) family proteins (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulators of oxidative stress responses. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons including genes associated withmore »thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites, revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea.« less