scholarly journals Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

2021 ◽  
Author(s):  
Yong Zi Tan ◽  
Yazan M Abbas ◽  
Jing Ze Wu ◽  
Di Wu ◽  
Geoffrey G Hesketh ◽  
...  
Keyword(s):  
Proton Pumps ◽  
Porcine Kidney ◽  
Subunit C ◽  
Binding Partners ◽  
Dauer Formation ◽  
Domain Protein ◽  
Endogenous Proteins ◽  
Α Helix ◽  
Rotary Catalysis ◽  
In Cells

V-ATPases are rotary proton pumps that serve as signaling hubs, with numerous proposed binding partners in cells. We used cryoEM to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK7 shows that mEAK7's TLDc domain, which is found in other proteins proposed to bind V-ATPase, interacts with V-ATPase's stator while its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, exogenous mEAK7 does not inhibit purified V-ATPase activity and mEAK7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Together, these results reveal how TLDc domains bind V-ATPases and suggest that V-ATPase binding proteins can form labile interactions that are sensitive to the enzyme's activity.

scholarly journals Reversible Thiol Oxidation Inhibits the Mitochondrial ATP Synthase in Xenopus laevis Oocytes

Antioxidants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 215 ◽  
Author(s):  
James Cobley ◽  
Anna Noble ◽  
Rachel Bessell ◽  
Matthew Guille ◽  
Holger Husi
Keyword(s):  
Xenopus Laevis ◽  
Atp Synthase ◽  
Xenopus Laevis Oocytes ◽  
Proton Pumps ◽  
Thiol Oxidation ◽  
Protein Thiol ◽  
Complex Iv ◽  
Subunit C ◽  
Catalyst Free ◽  
Mitochondrial Atp Synthase

Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min−1/mg−1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-α-F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min−1/mg−1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.

scholarly journals Split-TurboID enables contact-dependent proximity labeling in cells

2020 ◽  
Author(s):  
Kelvin F. Cho ◽  
Tess C. Branon ◽  
Sanjana Rajeev ◽  
Tanya Svinkina ◽  
Namrata D. Udeshi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interaction ◽  
Proteomic Analysis ◽  
Protein Protein Interaction ◽  
Conventional Fractionation ◽  
Contact Sites ◽  
Biochemical Fractionation ◽  
Versatile Tool ◽  
Endogenous Proteins ◽  
In Cells

AbstractProximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

scholarly journals Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Justin M Jenson ◽  
Jeremy A Ryan ◽  
Robert A Grant ◽  
Anthony Letai ◽  
Amy E Keating
Keyword(s):  
Small Molecules ◽  
Cancer Progression ◽  
Binding Mode ◽  
Mitochondrial Outer Membrane ◽  
Binding Partners ◽  
Peptide Binding Groove ◽  
Cell Permeable Peptide ◽  
Binding Groove ◽  
Resistance To Chemotherapy ◽  
In Cells

Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.

scholarly journals Ancient conserved domain protein-1 binds copper and modifies its retention in cells

2007 ◽  
Vol 0 (0) ◽  
pp. 070702072507004-??? ◽  
Author(s):  
Alexandra Alderton ◽  
Paul Davies ◽  
Katie Illman ◽  
David R. Brown
Keyword(s):  
Conserved Domain ◽  
Domain Protein ◽  
In Cells

scholarly journals Pro239Ser: A Novel Recessive Mutation of the Pit-1 Gene in Seven Middle Eastern Children with Growth Hormone, Prolactin, and Thyrotropin Deficiency1

1998 ◽  
Vol 83 (6) ◽  
pp. 2079-2083 ◽  
Author(s):  
Flavia Pernasetti ◽  
Robert D. G. Milner ◽  
Abdullah A. Z. Al Ashwal ◽  
Francis de Zegher ◽  
Viviana M. Chavez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Anterior Pituitary ◽  
Middle Eastern ◽  
Recessive Mutation ◽  
Specific Gene ◽  
Pituitary Development ◽  
Domain Protein ◽  
Α Helix ◽  
Tsh Deficiency

Pit-1, a member of the POU-homeo domain protein family, is one of the transcription factors responsible for anterior pituitary development and pituitary-specific gene expression. Here, we describe seven children with GH, PRL, and TSH deficiency from three, reportedly unrelated, Middle Eastern families, harboring a newly recognized Pro->Ser recessive mutation in codon 239 of the Pit-1 gene. The mutated residue is located at the beginning of the second α-helix of the POU-homeodomain and is strictly conserved among all POU proteins. The Pro239Ser mutant binds DNA normally but is unable to stimulate transcription.

scholarly journals Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

2021 ◽  
pp. mbc.E21-03-0156
Author(s):  
Stefano Sala ◽  
Patrick W. Oakes
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Lim Domain ◽  
Lim Domains ◽  
Lim Domain Protein ◽  
The Family ◽  
Domain Protein ◽  
Cytoskeletal Elements ◽  
In Cells

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals Mapping of a N-terminal α-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

2021 ◽  
Author(s):  
Poonam Kakade ◽  
Hina Ojha ◽  
Olawale Raimi ◽  
Andrew Shaw ◽  
Andrew Waddell ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Human Fibroblasts ◽  
Bioinformatic Analysis ◽  
Kinase Domain ◽  
Major Mechanism ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Α Helix ◽  
Hdx Ms ◽  
In Cells

PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal alpha-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

scholarly journals Pentatricopeptide repeat domain protein 1 lowers the levels of mitochondrial leucine tRNAs in cells

2009 ◽  
Vol 37 (17) ◽  
pp. 5859-5867 ◽  
Author(s):  
Oliver Rackham ◽  
Stefan M. K. Davies ◽  
Anne-Marie J. Shearwood ◽  
Kristina L. Hamilton ◽  
James Whelan ◽  
...  
Keyword(s):  
Pentatricopeptide Repeat ◽  
Repeat Domain ◽  
Domain Protein ◽  
In Cells

scholarly journals Identification of Miro as a mitochondrial receptor for myosin XIX

2018 ◽  
Author(s):  
Stefanie J. Oeding ◽  
Katarzyna Majstrowicz ◽  
Xiao-Ping Hu ◽  
Vera Schwarz ◽  
Angelika Freitag ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Specific Receptor ◽  
Tail Region ◽  
Gtpase Domain ◽  
Mitochondrial Movement ◽  
Binding Partners ◽  
Potential Binding ◽  
Mitochondrial Distribution ◽  
In Cells

ABSTRACTMitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein and along actin filaments by myosin. Myosin XIX (Myo19) associates with the outer mitochondrial membrane, but no specific receptor has been identified. Using proximity BioID labeling, we identified Miro-1 and Miro-2 as potential binding partners of Myo19. Interaction studies show that Miro-1 binds to a C-terminal fragment of the Myo19 tail region and that Miro recruits the Myo19 tail in vivo. This recruitment is regulated by the nucleotide-state of the N-terminal Rho-like GTPase domain of Miro. Notably, Myo19 protein stability in cells depends on its association with Miro. Finally, Myo19 regulates the subcellular distribution of mitochondria. Downregulation, as well as overexpression, of Myo19 induces perinuclear collapse of mitochondria, phenocopying the loss of kinesin KIF5 or its mitochondrial receptor Miro. These results suggest that Miro coordinates microtubule- and actin-based mitochondrial movement.

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