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1.
Plasmid ; 86: 14-25, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27381852

RESUMO

Horizontal gene transfer plays a profound role in bacterial evolution by propelling the rapid transfer of genes and gene cassettes. Integrative and conjugative elements (ICEs) are one important mechanism driving horizontal gene transfer. ICEs, also known as conjugative transposons, reside on the host chromosome but can excise to form a conjugative DNA circle that is capable of transfer to other cells. Analysis of the large number of completed bacterial genome sequences has revealed many previously unrecognized ICEs, including ICEBs1, found in the Gram-positive model bacterium Bacillus subtilis. The discovery of ICEBs1 in an organism with such an impressive array of molecular tools for genetics and molecular biology was fortuitous. Significant insights into ICE biology have resulted since its discovery <15years ago. In this review, we describe aspects of ICEBs1 biology, such as excision, conjugative transfer, and reintegration, likely to be conserved across many ICEs. We will also highlight some of the more unexpected aspects of ICEBs1 biology, such as its ability to undergo plasmid-like replication after excision and its ability to mobilize plasmids lacking dedicated mobilization functions. A molecular understanding of ICEBs1 has led to additional insights into signals and mechanisms that promote horizontal gene transfer and shape bacterial evolution.


Assuntos
Bacillus subtilis/genética , Conjugação Genética/genética , Replicação do DNA/fisiologia , Elementos de DNA Transponíveis/genética , Transferência Genética Horizontal/fisiologia , Plasmídeos/genética , DNA Bacteriano/genética , DNA Circular/genética
2.
Cancer Discov ; 14(2): 240-257, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37916956

RESUMO

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.


Assuntos
Neoplasias da Mama , Hiperinsulinismo , Humanos , Feminino , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Microscopia Crioeletrônica , Neoplasias da Mama/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/genética , Hiperinsulinismo/tratamento farmacológico , Hiperinsulinismo/genética , DNA
3.
Open Biochem J ; 12: 1-15, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29456767

RESUMO

BACKGROUND: Fumarase, a significant enzyme of energy metabolism, catalyzes the reversible hydration of fumarate to L-malate. Mutations in the FH gene, encoding human fumarase, are associated with fumarate hydratase deficiency (FHD) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Fumarase assembles into a homotetramer, with four active sites. Interestingly, residues from three of the four subunits within the homotetramer comprise each active site. Hence, any mutation affecting oligomerization is predicted to disrupt enzyme activity. METHODS: We constructed two variants of hexahistidine-tagged human recombinant fumarase, A308T and H318Y, associated with FHD and HLRCC, respectively. Both Ala308 and His318 lie within the fumarase intersubunit interface. We purified unmodified human fumarase and the two variants, and analyzed their enzymatic activities and oligomerization states in vitro. RESULTS: Both variants showed severely diminished fumarase activity. Steady-state kinetic analysis demonstrated that the variants were largely defective due to decreased turnover rate, while displaying Km values for L-malate similar to unmodified human recombinant fumarase. Blue native polyacrylamide gel electrophoresis and gel filtration experiments revealed that each variant had an altered oligomerization state, largely forming homodimers rather than homotetramers. CONCLUSION: We conclude that A308T and H318Y render human fumarase enzymatically inactive via defective oligomerization. Therefore, some forms of FHD and HLRCC can be linked to improperly folded quaternary structure.

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