scholarly journals MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

2019 ◽  
Author(s):  
Zhihao Wu ◽  
Ishaq Tantray ◽  
Junghyun Lim ◽  
Songjie Chen ◽  
Yu Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mitochondrial Dysfunction ◽  
Complex I ◽  
Mammalian Cells ◽  
Protein C ◽  
Stop Codon ◽  
Translation Termination ◽  
Disease Model ◽  
C Terminus ◽  
Translational Termination

SUMMARYMitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson’s disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

scholarly journals uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

2019 ◽  
Vol 47 (21) ◽  
pp. 11326-11343 ◽  
Author(s):  
Kristýna Poncová ◽  
Susan Wagner ◽  
Myrte Esmeralda Jansen ◽  
Petra Beznosková ◽  
Stanislava Gunišová ◽  
...  
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
C Terminus ◽  
Context Specific ◽  
Stop Codon Readthrough

Abstract Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

Subcellular localization of the Streptococcus mutans P1 protein C terminus

1999 ◽  
Vol 45 (6) ◽  
pp. 536-539 ◽  
Author(s):  
Mary K Homonylo-McGavin ◽  
Song F Lee ◽  
George H Bowden
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Cell Wall ◽  
Streptococcus Mutans ◽  
Polyclonal Antibody ◽  
Cell Walls ◽  
Protein C ◽  
Protein Localization ◽  
Subcellular Location ◽  
C Terminus

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.Key words: antigen P1, cell wall proteins, fusion proteins, peptidoglycan, protein localization.

scholarly journals Display of Heterologous Proteins on gp64null Baculovirus Virions and Enhanced Budding Mediated by a Vesicular Stomatitis Virus G-Stem Construct

2007 ◽  
Vol 82 (3) ◽  
pp. 1368-1377 ◽  
Author(s):  
Jian Zhou ◽  
Gary W. Blissard
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Heterologous Proteins ◽  
C Terminus ◽  
Influenza Virus Hemagglutinin ◽  
Severe Reduction ◽  
Vesicular Stomatitis ◽  
Ha Protein

ABSTRACT The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.

Calcium-Independent Detection and Isolation of Phosphatidylserine-Exposing Cells and Microparticles Using Recombinant Del-1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4224-4224
Author(s):  
Pavel Davizon ◽  
John Kulman ◽  
José A. López
Keyword(s):  
Flow Cytometry ◽  
Mammalian Cells ◽  
Protein C ◽  
Matrix Protein ◽  
Magnetic Beads ◽  
Annexin V ◽  
Extracellular Matrix Protein ◽  
Apoptotic Bodies ◽  
Embryonic Period ◽  
C Terminus

Abstract Abstract 4224 Del-1 is an extracellular matrix protein that is highly expressed during the embryonic period. It comprises, from N- to C-terminus, three epidermal growth factor (EGF) domains (containing an RGD motif in the second EGF domain) and two factor VIII-homologous C domains (C1C2 domain). After the embryonic period, hypoxia and vascular injury can increase Del-1 expression by endothelial cells and macrophages, promoting angiogenesis and phagocytosis of phosphatidylserine (PS)-exposing apoptotic bodies. This is possibly due to the capacity of Del-1 to bridge the interaction between apoptotic bodies and phagocytes. Del-1 binds to PS-exposing cells and membrane fragments through its C1C2 domains and to integrins of phagocytic cells through its RGD sequence, enhancing phagocytosis. We expressed in mammalian cells a Del-1 fragment composed of the C1 and C2 domains containing two human protein C tags and a biotin tag at the C-terminus. The recombinant fragment was purifed using monomeric avidin resin. The recombinant Del-1 C1C2 bound PS specifically on a commercial lipid array (PIP-strip, Echelon), a finding confirmed in a 96-well assay using immobilized lipids. The binding of C1C2 Del-1 to phosphatidylserine (PS) was consistent and stable when compared to the binding of commercially available annexin-V (biotinylated and PE-conjugated). We evaluated by flow cytometry the ability of Del-1 C1C2 to bind membrane-exposed PS. Apoptotic THP-1 cells were incubated with Del-1 C1C2 in the absence of calcium or with annexin V (2.5 mM calcium was present in all the buffers and through all washing steps). Protein binding was detected using a monoclonal anti-biotin-PE conjugated antibody. Neither the percentage of PS-positive cells nor the mean fluorescence intensity (MFI) was statistically different between Del-1 C1C2 and annexin V when samples were diluted with buffer (instead of washed) before flow cytometric analysis. However, both the percentage of PS-positive cells and MFI decreased by approximately 50% in the annexin V samples when the cells were washed after incubation. Cell-derived microparticles from outdated RBC units were also used to compare the utility of C1C2 Del-1 vs commercial PE-conjugated annexin V for the detection of microparticles by flow cytometry. We found a 1.4-fold average increase in the quantity of microparticles detected with C1C2 Del-1 when compared to annexin V. In addition, cell-derived microparticles can be isolated and concentrated from the Del-1 C1C2–treated sample using magnetic beads coupled to anti-protein C antibody. In conclusion, the C1C2 domain of Del-1 represents a powerful and sensitive tool for the detection and isolation of PS-exposing cells and microparticles. The calcium-independence of this reagent for PS simplifies the detection of apoptotic cells and microparticles in plasma. Moreover, the addition of multiple tags at the C-terminus allows the capture, labeling and enrichment of PS-specific microparticles by bridging C1C2-coated microparticles to magnetic beads. C1C2 Del-1 thus represents a powerful new tool for analysis in the fields of microparticle biology, thrombosis, and immunology. Disclosures: No relevant conflicts of interest to declare.

Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein

2001 ◽  
Vol 359 (3) ◽  
pp. 707-714 ◽  
Author(s):  
Dong CHENG ◽  
Rupalie L. MEEGALLA ◽  
Bokang HE ◽  
Debra A. CROMLEY ◽  
Jeffery T. BILLHEIMER ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Gel Filtration ◽  
Human Adipose Tissue ◽  
Cross Linking ◽  
Triacylglycerol Synthesis ◽  
Putative Active Site ◽  
Membrane Enzyme ◽  
C Terminus ◽  
Sds Page

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108kDa) and the tetramer (214kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.

scholarly journals Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis

2020 ◽  
Vol 295 (41) ◽  
pp. 14053-14064
Author(s):  
Ayodele Akintayo ◽  
Joshua Mayoral ◽  
Masahiro Asada ◽  
Jian Tang ◽  
Subha Sundaram ◽  
...  
Keyword(s):  
Amino Acids ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Mechanism Of Inhibition ◽  
C Terminus ◽  
Membrane Bound ◽  
Galanthus Nivalis ◽  
Glcnac Transferase

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

scholarly journals Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

2019 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

AbstractStop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion

2014 ◽  
Vol 25 (21) ◽  
pp. 3363-3374 ◽  
Author(s):  
Dan Dou ◽  
Diogo V. da Silva ◽  
Johan Nordholm ◽  
Hao Wang ◽  
Robert Daniels
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Mammalian Cells ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Membrane Insertion ◽  
Type Ii ◽  
C Terminus ◽  
Insertion Process ◽  
Positively Charged

Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the Nout-CinHA and M2 TMDs but not the Nin-CoutTMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (ΔGapp> 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ∼70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ∼100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

scholarly journals Mapping of the VP40-Binding Regions of the Nucleoprotein of Ebola Virus

2007 ◽  
Vol 81 (7) ◽  
pp. 3554-3562 ◽  
Author(s):  
Takeshi Noda ◽  
Shinji Watanabe ◽  
Hiroshi Sagara ◽  
Yoshihiro Kawaoka
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Assembly ◽  
Helical Structures ◽  
Antiviral Compounds ◽  
C Terminus ◽  
The Matrix ◽  
Ebola Virus Infection

ABSTRACT Expression of Ebola virus nucleoprotein (NP) in mammalian cells leads to the formation of helical structures, which serve as a scaffold for the nucleocapsid. We recently found that NP binding with the matrix protein VP40 is important for nucleocapsid incorporation into virions (T. Noda, H. Ebihara, Y. Muramoto, K. Fujii, A. Takada, H. Sagara, J. H. Kim, H. Kida, H. Feldmann, and Y. Kawaoka, PLoS Pathog. 2:e99, 2006). To identify the region(s) on the NP molecule required for VP40 binding, we examined the interaction of a series of NP deletion mutants with VP40 biochemically and ultrastructurally. We found that both termini of NP (amino acids 2 to 150 and 601 to 739) are essential for its interaction with VP40 and for its incorporation into virus-like particles (VLPs). We also found that the C terminus of NP is important for nucleocapsid incorporation into virions. Of interest is that the formation of NP helices, which involves the N-terminal 450 amino acids of NP, is dispensable for NP incorporation into VLPs. These findings enhance our understanding of Ebola virus assembly and in so doing move us closer to the identification of targets for the development of antiviral compounds to combat Ebola virus infection.

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