المؤلفون: Geetika Singh, Cristina D. Guibao, Jayaraman Seetharaman, Anup Aggarwal, Christy R. Grace, Dan E. McNamara, Sivaraja Vaithiyalingam, M. Brett Waddell, Tudor Moldoveanu

المصدر: Nature Communications, Vol 13, Iss 1, Pp 1-15 (2022)
Nature Communications

مصطلحات موضوعية: Multidisciplinary, Cell Death, Science, General Physics and Astronomy, Membrane Proteins, Apoptosis, General Chemistry, Crystallography, X-Ray, Ligands, General Biochemistry, Genetics and Molecular Biology, Article, Mitochondria, bcl-2 Homologous Antagonist-Killer Protein, Proto-Oncogene Proteins c-bcl-2, Liposomes, Humans, biological phenomena, cell phenomena, and immunity, BH3 Interacting Domain Death Agonist Protein, X-ray crystallography

الوصف: BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.
The authors show that the mechanism of BAK activation in mitochondrial apoptosis involves cooperation between direct activation by BH3-only protein BID and BAK autoactivation, providing a unifying basis for BAK triggering by BH3 ligands.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::eb8385f4f44c0480b152b55aa3c2761dTest
https://doaj.orgTest/article/948c7729db444ae0965cb207d8ea6853

المؤلفون: Barbara Jonchere, Jonathan Low, Michele Connelly, Liying Chi, Sergio C. Chai, R. Kiplin Guy, Kevin W. Freeman, Brandon Young, Jeanine E. Price, Mi-Kyung Yun, Marie Morfouace, Anang A. Shelat, Geoffrey Neale, Rachelle R. Olsen, Nagakumar Bharatham, Daniel Savic, Stephen W. White, P. Jake Slavish, Vincent A. Boyd, Lyudmila Tsurkan, Jun J. Yang, Nancy E. Martinez, Zhenmei Li, Martine F. Roussel, Philip M. Potter, M. Brett Waddell, William R. Shadrick, Richard E. Lee, Taosheng Chen, Sourav Das

المصدر: Cancer Res

مصطلحات موضوعية: 0301 basic medicine, Cancer Research, Antineoplastic Agents, Context (language use), Mice, SCID, Article, Proto-Oncogene Proteins c-myc, Mice, Structure-Activity Relationship, 03 medical and health sciences, 0302 clinical medicine, Protein Domains, Cell Line, Tumor, Neoplasms, Neuroblastoma, medicine, Animals, Humans, Child, Cytotoxicity, Oncogene, Chemistry, Cancer, medicine.disease, Xenograft Model Antitumor Assays, Pediatric cancer, Bromodomain, 030104 developmental biology, Oncology, Drug development, Drug Design, 030220 oncology & carcinogenesis, Cancer research, Female, Transcription Factors

الوصف: Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. Significance: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::86d687ae06b408a0a8a364651477a03fTest
https://doi.org/10.1158/0008-5472.can-19-3934Test

المؤلفون: Mi-Kyung Yun, Maksym Tsytlonok, Peter Tompa, Sivaraja Vaithiyalingam, Suren Felekyan, Claus A. M. Seidel, Aaron H. Phillips, Luigi I. Iconaru, Yuefeng Wang, Hugo Sanabria, Stephen W. White, M. Brett Waddell, Richard W. Kriwacki, Katherina Hemmen, Cheon-Gil Park

المصدر: Nature Communications, Vol 10, Iss 1, Pp 1-13 (2019)
Nature Communications

مصطلحات موضوعية: Threonine, 0301 basic medicine, Quantitative Biology - Subcellular Processes, Cell division, Cyclin A, Fusion Proteins, bcr-abl, General Physics and Astronomy, 02 engineering and technology, Crystallography, X-Ray, Quantitative Biology - Quantitative Methods, chemistry.chemical_compound, Phosphorylation, lcsh:Science, Quantitative Methods (q-bio.QM), Multidisciplinary, biology, Chemistry, 021001 nanoscience & nanotechnology, Recombinant Proteins, Cell biology, src-Family Kinases, 0210 nano-technology, Tyrosine kinase, Cell Division, Cyclin-Dependent Kinase Inhibitor p27, Protein Binding, Signal Transduction, Proto-oncogene tyrosine-protein kinase Src, Science, Molecular Dynamics Simulation, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Cyclin-dependent kinase, Subcellular Processes (q-bio.SC), Cyclin-Dependent Kinase 2, Cyclin-dependent kinase 2, Tyrosine phosphorylation, Biomolecules (q-bio.BM), General Chemistry, Protein Structure, Tertiary, 030104 developmental biology, Quantitative Biology - Biomolecules, FOS: Biological sciences, Proteolysis, Mutagenesis, Site-Directed, biology.protein, Tyrosine, lcsh:Q, Protein Processing, Post-Translational

الوصف: p27$^{Kip1}$ (p27) is an intrinsically disordered protein (IDP) that folds upon binding to cyclin-dependent kinase (Cdk)$/$cyclin complexes (e.g., Cdk2$/$cyclin A), inhibiting their catalytic activity and causing cell cycle arrest. However, cell division progresses when stably Cdk2$/$cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues $[$tyrosines 88 (Y88) and 74 (Y74)$]$ and a distal threonine residue $[$threonine 187 (T187)$]$. These events trigger ubiquitination and degradation of p27, fully activating Cdk2$/$cyclin A to drive cell division. Using an integrated approach comprising structural, biochemical, biophysical and single-molecule fluorescence methods, we show that Cdk2$/$cyclin A-bound p27 samples lowly-populated conformations that dynamically anticipate the sequential steps of this signaling cascade. "Dynamic anticipation" provides access to the non-receptor tyrosine kinases, BCR-ABL and Src, which sequentially phosphorylate Y88 and Y74 and promote intra-assembly phosphorylation (of p27) on distal T187. Tyrosine phosphorylation also allosterically relieves p27-dependent inhibition of substrate binding to Cdk2$/$cyclin A, a phenomenon we term "cross-complex allostery". Even when tightly bound to Cdk2$/$cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease, as exemplified by hyper-active BCR-ABL and Src in certain cancers.
35 pages, 5 figures, supporting information 37 pages

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::aebf5b582a25ff50bd576925e3ed1b6aTest
http://arxiv.org/abs/1812.07009Test

المؤلفون: Richard E. Lee, Stephen W. White, Zhenmei Li, M. Brett Waddell, Ying Zhao, Donald Bashford, Antonio M. Ferreira, Yinan Wu, Mi Kyung Yun

المصدر: Science. 335:1110-1114

مصطلحات موضوعية: Models, Molecular, Sulfamethoxazole, Protein Conformation, Yersinia pestis, Stereochemistry, Molecular Sequence Data, Parabens, DHPS, Biology, Crystallography, X-Ray, Article, chemistry.chemical_compound, Sulfathiazole, Protein structure, Catalytic Domain, parasitic diseases, Drug Resistance, Bacterial, 4-Aminobenzoic acid, Transferase, Magnesium, Amino Acid Sequence, Pterin, Sulfathiazoles, chemistry.chemical_classification, Dihydropteroate Synthase, Multidisciplinary, Mutagenesis, Anti-Bacterial Agents, Sulfonamide, Diphosphates, Models, Chemical, Biochemistry, chemistry, Bacillus anthracis, Biocatalysis, Crystallization, Dihydropteroate synthase, 4-Aminobenzoic Acid

الوصف: Sulfa's Crystal View The sulfonamide antibiotics (sulfa drugs) have been used to treat infections for over 70 years; however, emerging resistance has eroded their clinical utility. Sulfa drugs target dihydropteroate synthase, a key enzyme in the bacterial folate pathway. By performing the reaction in the crystalline form of the enzyme, Yun et al. (p. 1110 ) have characterized the key structural intermediates. In combining structural data with theoretical and mutagenesis studies, they propose a detailed mechanism for dihydropteroate synthase catalysis. By resolving this structure with a sulfa drug bound to the enzyme, they showed how inhibition occurred and indicated how resistance could emerge.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::972532e2c1adb13ccd74474a72d311baTest
https://doi.org/10.1126/science.1214641Test

المؤلفون: Steven N. Seyedin, Timothy Hoggard, Jiang Liu, Amanda Nourse, Min Zhuang, Michal Hammel, David M. Duda, Kevin P. White, J. Wade Harper, Helen Walden, Matthew F. Calabrese, Darcie J. Miller, Brenda A. Schulman, M. Brett Waddell

المصدر: Molecular Cell. 36(1):39-50

مصطلحات موضوعية: Models, Molecular, Co-Repressor Proteins, Recombinant Fusion Proteins, Ubiquitin-Protein Ligases, Computational biology, SPOP, Crystallography, X-Ray, Article, Histones, Protein structure, Consensus Sequence, Phosphoprotein Phosphatases, Animals, Drosophila Proteins, Humans, Protein Interaction Domains and Motifs, Amino Acid Sequence, Protein Structure, Quaternary, Molecular Biology, Adaptor Proteins, Signal Transducing, biology, Cullin Proteins, Ubiquitination, Nuclear Proteins, Cell Biology, Peptide Fragments, Chromatin, Ubiquitin ligase, DNA-Binding Proteins, Repressor Proteins, Drosophila melanogaster, Biochemistry, Mutation, biology.protein, Protein Multimerization, Cullin, Molecular Chaperones, Protein Binding, Transcription Factors

الوصف: In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC), and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call “3-box” due to its facilitating Cul3-binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3, and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ba3c3d9cbf9cfaab45e912fafb9471e2Test

المؤلفون: Daniel Hoagland, M. Brett Waddell, Richard E. Lee, Mi-Kyung Yun, Charles O. Rock, Gyanendra Kumar, Stephen W. White

المصدر: Bioorganicmedicinal chemistry. 22(7)

مصطلحات موضوعية: Models, Molecular, Stereochemistry, Clinical Biochemistry, Pharmaceutical Science, Crystallography, X-Ray, Biochemistry, Article, chemistry.chemical_compound, Structure-Activity Relationship, Drug Discovery, Structure–activity relationship, Hydroxymethyl, Pterin, Enzyme Inhibitors, Thioguanine, Molecular Biology, chemistry.chemical_classification, Dose-Response Relationship, Drug, Molecular Structure, Organic Chemistry, Antimicrobial, Enzyme, chemistry, Diphosphotransferases, Molecular Medicine, Structure based

الوصف: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::90f9921bdc40b87ae834f986df41d1ceTest
https://pubmed.ncbi.nlm.nih.gov/24613625Test

المؤلفون: M. Brett Waddell, Richard W. Kriwacki, Li Ou

المصدر: ACS chemical biology. 7(4)

مصطلحات موضوعية: Cyclin-dependent kinase 1, biology, Cyclin D, Cyclin A, Cell Cycle, Cyclin-Dependent Kinase 4, General Medicine, Cell Cycle Checkpoints, Cell cycle, Biochemistry, Cyclin-Dependent Kinases, Article, Cell biology, Cyclin-dependent kinase, Cyclins, biology.protein, Cyclin-dependent kinase complex, Molecular Medicine, Animals, Humans, Protein phosphorylation, Phosphorylation, Cyclin A2, Cyclin-Dependent Kinase Inhibitor p27

الوصف: p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3cb241f71b4facc2cd3f6723af9ab755Test
https://pubmed.ncbi.nlm.nih.gov/22276948Test

المؤلفون: David M. Duda, Laura A. Borg, Mary-Ann Bjornsti, M. Brett Waddell, Brenda A. Schulman, Robert C.A.M. van Waardenburg, Amanda Nourse, Sierra McGarity

مصطلحات موضوعية: Saccharomyces cerevisiae Proteins, Molecular Sequence Data, Molecular Conformation, SUMO binding, SUMO enzymes, Plasma protein binding, Saccharomyces cerevisiae, Biology, Ubiquitin-conjugating enzyme, Crystallography, X-Ray, Article, Ubiquitin, Structural Biology, Small Ubiquitin-Related Modifier Proteins, Catalytic Domain, Humans, Amino Acid Sequence, Cysteine, Binding site, Cloning, Molecular, Molecular Biology, Sequence Homology, Amino Acid, Effector, Proteins, Cell biology, Repressor Proteins, Biochemistry, Ubiquitin-Conjugating Enzymes, biology.protein, Protein Binding

الوصف: The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 A resolution crystal structure of a non-covalent Smt3p-Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the non-covalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds non-covalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the non-covalent Smt3p-Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p-Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p-Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that non-covalent ubiquitin-like protein-E2 complexes are conserved platforms, which function as parts of larger assemblies involved in many protein post-translational regulatory pathways.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c0130a7febb824b9e831dbd4b9b8596dTest
https://europepmc.org/articles/PMC1936411Test/