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1.
J Biol Chem ; 298(12): 102677, 2022 12.
Article in English | MEDLINE | ID: mdl-36336075

ABSTRACT

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and ß subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate ß subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:ß assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.


Subject(s)
Cytokines , Protein Disulfide-Isomerases , Humans , Interleukin-12 , Interleukin-23 , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Disulfide-Isomerases/metabolism , Protein Folding , Endoplasmic Reticulum
2.
Sci Rep ; 11(1): 11185, 2021 05 27.
Article in English | MEDLINE | ID: mdl-34045646

ABSTRACT

The human mitochondrial ClpXP protease complex (HsClpXP) has recently attracted major attention as a target for novel anti-cancer therapies. Despite its important role in disease progression, the cellular role of HsClpXP is poorly characterized and only few small molecule inhibitors have been reported. Herein, we screened previously established S. aureus ClpXP inhibitors against the related human protease complex and identified potent small molecules against human ClpXP. The hit compounds showed anti-cancer activity in a panoply of leukemia, liver and breast cancer cell lines. We found that the bacterial ClpXP inhibitor 334 impairs the electron transport chain (ETC), enhances the production of mitochondrial reactive oxygen species (mtROS) and thereby promotes protein carbonylation, aberrant proteostasis and apoptosis. In addition, 334 induces cell death in re-isolated patient-derived xenograft (PDX) leukemia cells, potentiates the effect of DNA-damaging cytostatics and re-sensitizes resistant cancers to chemotherapy in non-apoptotic doses.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Mitochondrial Proteins/antagonists & inhibitors , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Staphylococcus aureus
3.
ACS Chem Biol ; 15(1): 254-261, 2020 01 17.
Article in English | MEDLINE | ID: mdl-31825581

ABSTRACT

The pyridoxal 5'-phosphate-binding protein (PLPBP) is an evolutionarily conserved protein linked to pyridoxal 5'-phosphate-binding. Although mutations in PLPBP were shown to cause vitamin B6-dependent epilepsy, its cellular role and function remain elusive. We here report a detailed biochemical investigation of human PLPBP and its epilepsy-causing mutants by evaluating stability, cofactor binding, and oligomerization. In this context, chemical cross-linking combined with mass spectrometry unraveled an unexpected dimeric assembly of PLPBP. Furthermore, the interaction network of PLPBP was elucidated by chemical cross-linking paired with co-immunoprecipitation. A mass spectrometric analysis in a PLPBP knockout cell line resulted in distinct proteomic changes compared to wild type cells, including upregulation of several cytoskeleton- and cell division-associated proteins. Finally, transfection experiments with vitamin B6-dependent epilepsy-causing PLPBP variants indicate a potential role of PLPBP in cell division as well as proper muscle function. Taken together, our studies on the structure and cellular role of human PLPBP enable a better understanding of the physiological and pathological mechanism of this important protein.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Proteomics/methods , Cell Line , Cross-Linking Reagents/chemistry , Epilepsy/metabolism , Gene Expression Regulation , Humans , Immunoprecipitation , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Solubility , Tandem Mass Spectrometry , Vitamin B 6/chemistry
4.
Cell Chem Biol ; 26(10): 1461-1468.e7, 2019 10 17.
Article in English | MEDLINE | ID: mdl-31447350

ABSTRACT

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor that catalyzes a plethora of chemical transformations within a cell. Although many human PLP-dependent enzymes (PLP-DEs) with crucial physiological and pathological roles are known, a global method enabling their cellular profiling is lacking. Here, we demonstrate the utility of a cofactor probe for the identification of human PLP-binding proteins in living cells. Striking selectivity of human pyridoxal kinase led to a customized labeling strategy covering a large fraction of known PLP-binding proteins across various cancer-derived cell lines. Labeling intensities of some PLP-DEs varied depending on the cell type while the overall protein expression levels of these proteins remained constant. In addition, we applied the methodology for in situ screening of PLP-antagonists and unraveled known binders as well as unknown off-targets. Taken together, our proteome-wide method to study PLP-DEs in human cancer-derived cells enables global understanding of the interactome of this important cofactor.


Subject(s)
Carrier Proteins/metabolism , Proteome/metabolism , Carrier Proteins/antagonists & inhibitors , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , K562 Cells , Models, Molecular , Molecular Structure , Penicillamine/pharmacology , Proteome/antagonists & inhibitors
5.
Cell Chem Biol ; 26(1): 48-59.e7, 2019 01 17.
Article in English | MEDLINE | ID: mdl-30415967

ABSTRACT

Detection of dynamic protein-protein interactions within complexes and networks remains a challenging task. Here, we show by the example of the proteolytic ClpXP complex the utility of combined chemical cross-linking and mass spectrometry (XL-MS) to map interactions within ClpP and ClpX as well as across the enigmatic ClpX hexamer-ClpP heptamer interface. A few hot-spot lysines located in signature loops in ClpX were shown to be in proximity to several structural regions of ClpP providing an initial draft of the ClpX-ClpP interaction. Application of XL-MS further confirmed that Listeria monocytogenes ClpX interacts with the heterooligomeric ClpP1/2 complex solely via the ClpP2 apical site. Moreover, cellular interaction networks of human and bacterial proteases were elucidated via in situ chemical cross-linking followed by an antibody-based pull-down against ClpP. A subsequent mass spectrometric analysis demonstrated an up to 3-fold higher coverage compared with co-immunoprecipitation without cross-linker revealing unprecedented insight into intracellular ClpXP networks.


Subject(s)
Cross-Linking Reagents/chemistry , Endopeptidase Clp/chemistry , Endopeptidase Clp/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Protein Interaction Mapping , Protein Interaction Maps , Cross-Linking Reagents/chemical synthesis , Female , Hep G2 Cells , Humans , K562 Cells , Male , Mass Spectrometry , Proteolysis
6.
J Proteome Res ; 16(3): 1180-1192, 2017 03 03.
Article in English | MEDLINE | ID: mdl-28186774

ABSTRACT

ß-Lactones have recently been introduced as the first selective ClpP inhibitors that attenuate virulence of both sensitive Staphylococcus aureus and multiresistant strains (MRSA). Although previous knockout studies showed that ClpP is essential for S. aureus alpha-toxin production, a link between ß-lactone inhibition and molecular virulence mechanisms has been lacking so far. We here perform a chemical-proteomic approach to elucidate antivirulence pathways. First, we demonstrate by gel-free activity-based protein profiling that ClpP is the predominant target of ß-lactones. Only a few off-targets were discovered, which, unlike ClpP, were not involved in the reduction of alpha-toxin expression. Second, in-depth mechanistic insight was provided by a full proteomic comparison between lactone treated and untreated S. aureus cells. Quantitative mass-spectrometric analysis revealed increased repressor of toxin (Rot) levels and a corresponding down-regulation of α-toxin, providing the first direct connection between the lactone-dependent phenotype and a corresponding cellular mechanism. By building up a quantitative virulence regulation network, we visualize the impact of ClpP inhibition in a systems biology context. Interestingly, a lack of in vitro Rot degradation by either ClpXP or ClpCP calls either for a proteolysis mechanism with yet unknown adaptor proteins or for an indirect mode of action that may involve ClpX-mediated RNA signaling and feedback circuits.


Subject(s)
Lactones/pharmacology , Staphylococcus aureus/pathogenicity , Virulence/drug effects , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Endopeptidase Clp/drug effects , Gene Expression Regulation, Bacterial/drug effects , Proteomics , Repressor Proteins/analysis , Systems Biology
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