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1.
Nat Chem ; 16(10): 1592-1604, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39138346

RESUMO

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds-termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.


Assuntos
Acrilamida , Proteômica , Humanos , Acrilamida/química , Estereoisomerismo , Triptaminas/química , Triptaminas/farmacologia , Proteoma/metabolismo , Linhagem Celular Tumoral , Ligação Proteica , Carbolinas
2.
J Am Chem Soc ; 146(15): 10393-10406, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38569115

RESUMO

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.


Assuntos
Acrilamida , Diterpenos , Fenantrenos , Humanos , Cisteína/química , Proteômica , Compostos de Epóxi
3.
J Exp Bot ; 74(5): 1705-1722, 2023 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-36576197

RESUMO

J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/metabolismo , Filogenia , Agregados Proteicos
4.
Viruses ; 14(10)2022 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-36298715

RESUMO

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core "prion fragmentation machinery" includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These "polyQX" PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1's known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein-protein interactions with individual yeast prion-forming proteins.


Assuntos
Príons , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Príons/química , Proteínas de Saccharomyces cerevisiae/química , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Amiloide/química , Proteínas Amiloidogênicas/metabolismo , Aminoácidos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo
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