Effective degradation of EGFRL858R+T790M mutant proteins by CRBN-based PROTACs through both proteosome and autophagy/lysosome degradation systems

Abstract Numerous inhibitors of tyrosine-protein kinase KIT, a receptor tyrosine kinase, have been explored as a viable therapy for the treatment of gastrointestinal stromal tumor (GIST). However, drug resistance due to acquired mutations in KIT makes these drugs almost useless. The present study was designed to screen the novel inhibitors against the activity of the KIT mutants through pharmacophore modeling and molecular docking. The best two pharmacophore models were established using the KIT mutants’ crystal complexes and were used to screen the new compounds with possible KIT inhibitory activity against both activation loop and ATP-binding mutants. As a result, two compounds were identified as potential candidates from the virtual screening, which satisfied the potential binding capabilities, molecular modeling characteristics, and predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. Further molecular docking simulations showed that two compounds made strong hydrogen bond interaction with different KIT mutant proteins. Our results indicated that pharmacophore models based on the receptor–ligand complex had excellent ability to screen KIT inhibitors, and two compounds may have the potential to develop further as the future KIT inhibitors for GIST treatment.

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Cytosolic Ribosomal Mutations That Abolish Accumulation of Circular Intron in the Mitochondria Without Preventing Senescence of Podospora anserina

Genetics ◽

10.1093/genetics/145.3.697 ◽

1997 ◽

Vol 145 (3) ◽

pp. 697-705 ◽

Cited By ~ 1

Author(s):

Philippe Silar ◽

France Koll ◽

Michèle Rossignol

Keyword(s):

Mitochondrial Dna ◽

Ribosomal Proteins ◽

Podospora Anserina ◽

Cox1 Gene ◽

Accuracy Control ◽

Additional Function ◽

Mutant Proteins ◽

Double Stranded Dna ◽

Dna Modifications

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNAα (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron α) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNAα seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron α plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.

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Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽

10.1093/genetics/163.1.91 ◽

2003 ◽

Vol 163 (1) ◽

pp. 91-101 ◽

Cited By ~ 1

Author(s):

Erin N Asleson ◽

Dennis M Livingston

Keyword(s):

Half Life ◽

Translational Regulation ◽

Cellular Level ◽

Missense Mutations ◽

Wild Type ◽

Mutant Proteins ◽

Gal1 Promoter ◽

Rad52 Protein ◽

The Stability ◽

Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Evidence that glutamic acid 49 of tryptophan synthase alpha subunit is a catalytic residue. Inactive mutant proteins substituted at position 49 bind ligands and transmit ligand-dependent to the beta subunit.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68348-6 ◽

1988 ◽

Vol 263 (18) ◽

pp. 8611-8614 ◽

Cited By ~ 4

Author(s):

E W Miles ◽

P McPhie ◽

K Yutani

Keyword(s):

Glutamic Acid ◽

Beta Subunit ◽

Alpha Subunit ◽

Catalytic Residue ◽

Tryptophan Synthase ◽

Mutant Proteins

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Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

Cell Death and Disease ◽

10.1038/s41419-021-03657-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Seung Won Choi ◽

Yeri Lee ◽

Kayoung Shin ◽

Harim Koo ◽

Donggeon Kim ◽

...

Keyword(s):

Functional Properties ◽

Therapeutic Target ◽

Medical Center ◽

Dominant Negative ◽

The Cancer Genome Atlas ◽

Dominant Negative Effect ◽

Cell Periphery ◽

Missense Mutations ◽

Phosphatase Domain ◽

Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

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