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1.
Cell ; 186(16): 3443-3459.e24, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37480851

RESUMO

Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.


Assuntos
Chaperonina com TCP-1 , Ubiquitina-Proteína Ligases , Chaperonina com TCP-1/química , Ubiquitina-Proteína Ligases/genética , Humanos
2.
Mol Cell ; 83(13): 2290-2302.e13, 2023 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-37295431

RESUMO

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.


Assuntos
Ribossomos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Microtúbulos/metabolismo , Homeostase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA , Proteínas de Transporte/metabolismo , Fatores de Transcrição/metabolismo
3.
Nature ; 623(7988): 842-852, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37853127

RESUMO

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.


Assuntos
Substâncias Macromoleculares , Proteínas , Solventes , Termodinâmica , Água , Morte Celular , Citosol/química , Citosol/metabolismo , Homeostase , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Concentração Osmolar , Pressão , Proteínas/química , Proteínas/metabolismo , Solventes/química , Solventes/metabolismo , Temperatura , Fatores de Tempo , Água/química , Água/metabolismo
4.
EMBO J ; 41(11): e109985, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35466425

RESUMO

Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.


Assuntos
Piperidinas , Quinazolinonas , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fosforilação , Piperidinas/farmacologia , Prolina/metabolismo , Biossíntese de Proteínas , Quinazolinonas/farmacologia
5.
PLoS Pathog ; 19(4): e1011177, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37058467

RESUMO

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE1 subunits within the pilus are arranged in a zigzag architecture, containing an N-terminal donor ß-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, with comparatively weaker interactions at the rest of the inter-subunit interface. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which might facilitate their role in promoting cellular attachment. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili, providing a structural basis for understanding their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.


Assuntos
Fímbrias Bacterianas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Fímbrias Bacterianas/metabolismo , Biofilmes , Adesinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Fímbrias/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(31): e2202080119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35901214

RESUMO

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Pró-Proteína Convertase 9 , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Fenilbutiratos , Pró-Proteína Convertase 9/metabolismo , Mapeamento de Interação de Proteínas , Transporte Proteico , Via Secretória , Proteínas de Transporte Vesicular/metabolismo
8.
Int J Mol Sci ; 25(16)2024 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-39201434

RESUMO

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.


Assuntos
Coenzima A , Simulação de Acoplamento Molecular , Estresse Oxidativo , Proteínas Quinases S6 Ribossômicas 70-kDa , Humanos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Coenzima A/metabolismo , Fosforilação , Ligação Proteica , Sítios de Ligação , Cisteína/metabolismo , Simulação de Dinâmica Molecular
9.
J Cell Sci ; 132(2)2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30617109

RESUMO

Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.


Assuntos
Dictyostelium/enzimologia , Pinocitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Protozoários/metabolismo , Dictyostelium/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Protozoários/genética
10.
Biochem J ; 475(11): 1909-1937, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29626155

RESUMO

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.


Assuntos
Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Coenzima A/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Coenzima A/genética , Diamida/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Oxirredução , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
11.
Biochem J ; 474(14): 2489-2508, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28341808

RESUMO

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.


Assuntos
Coenzima A/metabolismo , Proteínas/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Miocárdio/metabolismo , Especificidade de Órgãos , Oxirredução , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Coelhos , Ratos Sprague-Dawley , Compostos de Sulfidrila/metabolismo
12.
Chembiochem ; 17(12): 1107-10, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-26992063

RESUMO

Nanoscale objects of increasing complexity can be constructed from DNA or RNA. However, the scope of potential applications could be enhanced by expanding beyond the moderate chemical diversity of natural nucleic acids. Here, we explore the construction of nano-objects made entirely from alternative building blocks: synthetic genetic polymers not found in nature, also called xeno nucleic acids (XNAs). Specifically, we describe assembly of 70 kDa tetrahedra elaborated in four different XNA chemistries (2'-fluro-2'-deoxy-ribofuranose nucleic acid (2'F-RNA), 2'-fluoroarabino nucleic acids (FANA), hexitol nucleic acids (HNA), and cyclohexene nucleic acids (CeNA)), as well as mixed designs, and a ∼600 kDa all-FANA octahedron, visualised by electron microscopy. Our results extend the chemical scope for programmable nanostructure assembly, with implications for the design of nano-objects and materials with an expanded range of structural and physicochemical properties, including enhanced biostability.


Assuntos
Nanoestruturas/química , Polímeros/química , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia Eletrônica de Transmissão , Ácidos Nucleicos/química
13.
Sci Adv ; 10(13): eadl0608, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38552021

RESUMO

The Golgi-localized golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23. The amino-terminal domain of TBC1D23 binds to the golgins, and the carboxyl-terminal domain of TBC1D23 captures the vesicles, but how it recognizes specific vesicles was unclear. A search for binding partners of the carboxyl-terminal domain unexpectedly revealed direct binding to carboxypeptidase D and syntaxin-16, known cargo proteins of the captured vesicles. Binding is via a threonine-leucine-tyrosine (TLY) sequence present in both proteins next to an acidic cluster. A crystal structure reveals how this acidic TLY motif binds to TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the carboxyl-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle.


Assuntos
Complexo de Golgi , Proteínas de Membrana , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Golgi/metabolismo , Transporte Biológico , Endossomos/metabolismo , Ligação Proteica
14.
Antioxidants (Basel) ; 12(4)2023 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-37107313

RESUMO

Coenzyme A (CoA) is an important cellular metabolite that is critical for metabolic processes and the regulation of gene expression. Recent discovery of the antioxidant function of CoA has highlighted its protective role that leads to the formation of a mixed disulfide bond with protein cysteines, which is termed protein CoAlation. To date, more than 2000 CoAlated bacterial and mammalian proteins have been identified in cellular responses to oxidative stress, with the majority being involved in metabolic pathways (60%). Studies have shown that protein CoAlation is a widespread post-translational modification which modulates the activity and conformation of the modified proteins. The induction of protein CoAlation by oxidative stress was found to be rapidly reversed after the removal of oxidizing agents from the medium of cultured cells. In this study, we developed an enzyme-linked immunosorbent assay (ELISA)-based deCoAlation assay to detect deCoAlation activity from Bacillus subtilis and Bacillus megaterium lysates. We then used a combination of ELISA-based assay and purification strategies to show that deCoAlation is an enzyme-driven mechanism. Using mass-spectrometry and deCoAlation assays, we identified B. subtilis YtpP (thioredoxin-like protein) and thioredoxin A (TrxA) as enzymes that can remove CoA from different substrates. With mutagenesis studies, we identified YtpP and TrxA catalytic cysteine residues and proposed a possible deCoAlation mechanism for CoAlated methionine sulfoxide reducatse A (MsrA) and peroxiredoxin 5 (PRDX5) proteins, which results in the release of both CoA and the reduced form of MsrA or PRDX5. Overall, this paper reveals the deCoAlation activity of YtpP and TrxA and opens doors to future studies on the CoA-mediated redox regulation of CoAlated proteins under various cellular stress conditions.

15.
Sci Adv ; 9(34): eadi4148, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37624885

RESUMO

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.


Assuntos
Nucleossomos , Telômero , Animais , Humanos , Cromatina , Cromossomos de Mamíferos , Microscopia Crioeletrônica , Mamíferos , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo
16.
Nat Chem ; 15(1): 91-100, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36229679

RESUMO

Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control 'gate' in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2'-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2'OMe-RNA (2'OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and ß-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2'OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2'-modified RNAs-used in several approved nucleic acid therapeutics-for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.


Assuntos
RNA , Fator A de Crescimento do Endotélio Vascular , RNA/química , Oligorribonucleotídeos , RNA Mensageiro
17.
Nat Chem ; 14(11): 1295-1305, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36064973

RESUMO

Nucleic-acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical applications. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2'-deoxy-2'-fluoro-ß-D-arabino nucleic acid (FANA). FR6_1 overcomes the activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (>5 kb) BRAF and KRAS mRNAs at physiological Mg2+ concentrations with allelic selectivity for tumour-associated (BRAF V600E and KRAS G12D) mutations. Phosphorothioate-FANA modification enhances FR6_1 biostability and enables rapid KRAS mRNA knockdown in cultured human adenocarcinoma cells with a G12D-allele-specific component provided by in vivo XNAzyme cleavage activity. These results provide a starting point for the development of improved gene-silencing agents based on FANA or other XNA chemistries.


Assuntos
Ácidos Nucleicos , Proteínas Proto-Oncogênicas B-raf , Humanos , Alelos , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA , Inativação Gênica
18.
Antioxidants (Basel) ; 11(7)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35883853

RESUMO

Coenzyme A (CoA) is a key cellular metabolite known for its diverse functions in metabolism and regulation of gene expression. CoA was recently shown to play an important antioxidant role under various cellular stress conditions by forming a disulfide bond with proteins, termed CoAlation. Using anti-CoA antibodies and liquid chromatography tandem mass spectrometry (LC-MS/MS) methodologies, CoAlated proteins were identified from various organisms/tissues/cell-lines under stress conditions. In this study, we integrated currently known CoAlated proteins into mammalian and bacterial datasets (CoAlomes), resulting in a total of 2093 CoAlated proteins (2862 CoAlation sites). Functional classification of these proteins showed that CoAlation is widespread among proteins involved in cellular metabolism, stress response and protein synthesis. Using 35 published CoAlated protein structures, we studied the stabilization interactions of each CoA segment (adenosine diphosphate (ADP) moiety and pantetheine tail) within the microenvironment of the modified cysteines. Alternating polar-non-polar residues, positively charged residues and hydrophobic interactions mainly stabilize the pantetheine tail, phosphate groups and the ADP moiety, respectively. A flexible nature of CoA is observed in examined structures, allowing it to adapt its conformation through interactions with residues surrounding the CoAlation site. Based on these findings, we propose three modes of CoA binding to proteins. Overall, this study summarizes currently available knowledge on CoAlated proteins, their functional distribution and CoA-protein stabilization interactions.

19.
J Biol Chem ; 285(26): 19927-34, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20430892

RESUMO

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFkappaB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP(6)). We extend the applicability of our results to show that IP(6) also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP(6)-induced conformational change in AvrA suggests that IP(6) acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP(6); once injected into mammalian cells by the pathogen these toxins bind host cell IP(6), are activated, and deregulate the MAPK and NFkappaB signaling pathways thereby subverting innate immunity.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Fítico/metabolismo , Yersinia/enzimologia , Acetiltransferases/química , Acetiltransferases/genética , Regulação Alostérica , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Citosol/metabolismo , Citosol/microbiologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Ácido Fítico/isolamento & purificação , Plasmídeos/genética , Conformação Proteica , Yersinia/fisiologia
20.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
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