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1.
Biochemistry ; 63(3): 251-263, 2024 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-38243804

RESUMEN

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.


Asunto(s)
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Escherichia coli/química , Sitios de Unión , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Péptidos/metabolismo , Unión Proteica
2.
J Am Chem Soc ; 146(30): 20845-20856, 2024 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-39041457

RESUMEN

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.


Asunto(s)
Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 10/química , Escherichia coli/efectos de los fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/química , Benzoxazoles/química , Benzoxazoles/farmacología , Benzoxazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química
3.
Redox Biol ; 65: 102839, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37573837

RESUMEN

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.


Asunto(s)
Proteínas Portadoras , Factor 2 Relacionado con NF-E2 , Humanos , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Retroalimentación , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo
4.
J Med Chem ; 66(1): 677-694, 2023 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-36516003

RESUMEN

A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8, was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.


Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacología , Chaperonas Moleculares/química , Proteínas HSP70 de Choque Térmico , Estrés del Retículo Endoplásmico , Isoformas de Proteínas
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