RESUMO
Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to -1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein-DNA and protein-protein interactions in biological processes from replication to ubiquitin signalling.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.
Assuntos
Mitose , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Linhagem Celular Tumoral , Instabilidade Cromossômica , Citoproteção , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Mutações Sintéticas LetaisRESUMO
Methylation of a conserved lysine in C-terminal domain of the molecular chaperone Hsp90 was shown previously to affect its in vivo function. However, the underlying mechanism remained elusive. Through a combined experimental and computational approach, this study shows that this site is very sensitive to sidechain modifications and crucial for Hsp90 activity in vitro and in vivo. Our results demonstrate that this particular lysine serves as a switch point for the regulation of Hsp90 functions by influencing its conformational cycle, ATPase activity, co-chaperone regulation, and client activation of yeast and human Hsp90. Incorporation of the methylated lysine via genetic code expansion specifically shows that upon modification, the conformational cycle of Hsp90 is altered. Molecular dynamics simulations including the methylated lysine suggest specific conformational changes that are propagated through Hsp90. Thus, methylation of the C-terminal lysine allows a precise allosteric tuning of Hsp90 activity via long distances.
Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Lisina/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Lisina/genética , Metilação , Simulação de Dinâmica Molecular , Mutação/genética , Nucleotídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Inverse electron-demand Diels-Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light-triggered version of iEDDAC (photo-iEDDAC) is presented that confers spatio-temporal control to bioorthogonal labeling inâ vitro and inâ cellulo. A cyclopropenone-caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo-DMBO) was designed that is unreactive towards tetrazines before light-activation, but engages in iEDDAC after irradiation at 365â nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site-specific incorporation of tetrazine-containing amino acids into proteins in living cells. Inâ situ light activation of photo-DMBO conjugates allows labeling of tetrazine-modified proteins in living E.â coli. This allows proteins in living cells to be modified in a spatio-temporally controlled manner and may be extended to photo-induced and site-specific protein labeling in animals.