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1.
bioRxiv ; 2024 Jul 13.
Article in English | MEDLINE | ID: mdl-39026748

ABSTRACT

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degraders function by proximity induction, and directly bridge an E3 ligase with the target to be degraded. However, many proteins might not be addressable via proximity-based degraders, and other challenges, such as resistance acquisition, exist. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs induce a unique conformational change and, thereby, boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2 KLHDC3 , which we identified to also mediate native IDO1 degradation. Therefore, iDegs supercharge the native proteolytic pathway of IDO1, rendering this mechanism of action distinct from traditional degrader approaches involving proteolysis-targeting chimeras (PROTACs) or molecular-glue degraders (MGDs). In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and should also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

2.
Chembiochem ; 24(24): e202300579, 2023 12 14.
Article in English | MEDLINE | ID: mdl-37869939

ABSTRACT

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.


Subject(s)
Autophagy , Lysosomes , Biological Transport , Galectin 3 , Endosomal Sorting Complexes Required for Transport/metabolism
3.
Angew Chem Int Ed Engl ; 62(21): e202301955, 2023 05 15.
Article in English | MEDLINE | ID: mdl-36929571

ABSTRACT

Oxindoles and iso-oxindoles are natural product-derived scaffolds that provide inspiration for the design and synthesis of novel biologically relevant compound classes. Notably, the spirocyclic connection of oxindoles with iso-oxindoles has not been explored by nature but promises to provide structurally related compounds endowed with novel bioactivity. Therefore, methods for their efficient synthesis and the conclusive discovery of their cellular targets are highly desirable. We describe a selective RhIII -catalyzed scaffold-divergent synthesis of spirooxindole-isooxindoles and spirooxindole-oxindoles from differently protected diazooxindoles and N-pivaloyloxy aryl amides which includes a functional group-controlled Lossen rearrangement as key step. Unbiased morphological profiling of a corresponding compound collection in the Cell Painting assay efficiently identified the mitotic kinesin Eg5 as the cellular target of the spirooxindoles, defining a unique Eg5 inhibitor chemotype.


Subject(s)
Kinesins , Oxindoles
4.
Biol Chem ; 389(2): 179-87, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18163890

ABSTRACT

Abstract Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha) is down-regulated in RCC and we have shown that HNF4 alpha inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4 alpha we analyzed the whole human expression profile in HEK293 cells upon HNF4 alpha induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4 alpha-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4 alpha. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4 alpha on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4 alpha target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4 alpha orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4 alpha could contribute to the progression of kidney cancer.


Subject(s)
Cell Proliferation , Gene Expression Regulation , Hepatocyte Nuclear Factor 4/physiology , Kidney/cytology , Cell Line , Down-Regulation , Gene Expression Profiling , Gene Regulatory Networks , Hepatocyte Nuclear Factor 4/genetics , Humans , Kidney Neoplasms/etiology , Kidney Neoplasms/pathology
5.
Biol Chem ; 388(1): 91-106, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17214554

ABSTRACT

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor expressed in many cell types, including pancreatic beta-cells. Mutations in the HNF4alpha gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by beta-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4alpha2 or HNF4alpha8 into the rat beta-cell line INS-1. Upon tetracycline-induced expression, both HNF4alpha isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4alpha2 compared to HNF4alpha8, and suggests that both isoforms regulate largely the same set of genes, with HNF4alpha2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4alpha may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.


Subject(s)
Apoptosis/physiology , Cell Proliferation , Hepatocyte Nuclear Factor 4/physiology , Insulin-Secreting Cells/metabolism , Animals , Apoptosis/genetics , Blotting, Western , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Hepatocyte Nuclear Factor 4/genetics , Humans , Insulin-Secreting Cells/cytology , Mutation , Oligonucleotide Array Sequence Analysis , Protein Isoforms/genetics , Protein Isoforms/physiology , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tetracycline/pharmacology , Time Factors
6.
Biochim Biophys Acta ; 1731(3): 179-90, 2005 Dec 20.
Article in English | MEDLINE | ID: mdl-16297991

ABSTRACT

Hepatocyte nuclear factor 1beta (HNF1beta, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1beta overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1beta target genes whose activity may be deregulated in human patients, we established a human embryonic kidney cell line (HEK293) expressing HNF1beta conditionally. Using Flp recombinase, we introduced wild type or mutated HNF1beta at a defined chromosomal position allowing a most reproducible induction of the HNF1beta derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1beta-regulated genes. By an identical approach, we identified that the related transcription factor HNF1alpha (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1beta target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1beta target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1beta overexpression in the ovarian cancer participates in the altered expression pattern.


Subject(s)
Hepatocyte Nuclear Factor 1-alpha/metabolism , Hepatocyte Nuclear Factor 1-beta/metabolism , Kidney/cytology , Transcription Factors/genetics , Angiotensin-Converting Enzyme 2 , Base Sequence , Carboxypeptidases/metabolism , DNA Nucleotidyltransferases/genetics , Gene Expression Regulation , Hepatocyte Nuclear Factor 1-alpha/physiology , Hepatocyte Nuclear Factor 1-beta/physiology , Humans , Kidney/metabolism , Models, Genetic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis/methods , Osteopontin , Peptidyl-Dipeptidase A , Promoter Regions, Genetic , Sialoglycoproteins/metabolism , Tetracycline/metabolism , Tetracycline/pharmacology , Transcription Factors/metabolism , Transgenes
7.
Oncogene ; 24(42): 6418-31, 2005 Sep 22.
Article in English | MEDLINE | ID: mdl-16007190

ABSTRACT

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic beta cells. Although HNF4alpha is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4alpha-regulated genes in the kidney cells. The abundance and activity of HNF4alpha are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4alpha in renal cells. To determine the potential role of HNF4alpha in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4alpha. Expression of wild-type HNF4alpha but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4alpha. Using gene expression profiling by microarrays, we determined genes regulated by HNF4alpha. Interestingly, many of the genes regulated by HNF4alpha have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4alpha in RCC.


Subject(s)
Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Division/physiology , DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic/physiology , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Kidney/pathology , Phosphoproteins/physiology , Transcription Factors/physiology , Base Sequence , Carcinoma, Renal Cell/metabolism , Cell Line , DNA Primers , Hepatocyte Nuclear Factor 4 , Humans , Kidney Neoplasms/metabolism , Microfilament Proteins/metabolism , Oligonucleotide Array Sequence Analysis , WT1 Proteins/metabolism
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