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Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase

ABSTRACT: The eight-subunit COP9 signalosome (CSN complex), controls the exchange of E3 ubiquitin cullin RING ligase receptors through its deneddylase activity, providing specificity to eukaryotic protein degradation. The conserved eight-subunit CSN complex is required for multicellular development of the filamentous fungus Aspergillus nidulans. The human, as well as the fungal CSN complex assembles through a heptameric subcomplex (pre-CSN complex) that is activated by the integration of the eighth subunit, the catalytic CsnE/5 deneddylase. The focus of this work was to unveil the assembly of the native fungal pre-CSN complex via combined genetic and biochemical approaches. Interactomes of various functional GFP-Csn subunit fusions, produced in pre-CSN deficient fungal double csn subunit gene deletion strains, were compared by affinity purifications coupled to mass spectrometry analyses. With this approach, two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates, namely CsnA/1-C/3-H/8 and CsnD/4-F/6-G/7 that form independently of CsnB/2 that connects the heterotrimers to a heptameric subcomplex and enables subsequent integration of CsnE/5, yielding in the enzymatically active CSN holocomplex. Surveillance mechanisms control accurate Csn subunit abundances and correct cellular localization for sequential assembly, since deprivation of Csn subunits change the abundance and location of the remaining Csn subunits.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Emericella Nidulans (strain Fgsc A4 / Atcc 38163 / Cbs 112.46 / Nrrl 194 / M139) (aspergillus Nidulans)

SUBMITTER: Oliver Valerius

LAB HEAD: Gerhard H. Braus

PROVIDER: PXD043205 | Pride | 2023-08-10

REPOSITORIES: Pride

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Project description:Senolytic drugs are designed to selectively clear senescent cells (SnCs) that accumulate with injury or aging. In a mouse model of osteoarthritis (OA), senolysis yields a pro-regenerative response. However, recent research studies showed that the therapeutic benefit is reduced in aged mice. Increased oxidative stress (redox) mediates metabolic dysregulation and accumulation of posttranslational modifications (PTM) on proteins from the cartilage and is one of the major hallmarks of advanced age. This research investigated whether the senolytic treatment differentially affects oxidative load in the joints from young and aged animals. We employed label-free quantitative (LFQ) analysis of changes in the protein expression profiles and associated carbonylated PTMs in the extracted joint proteins. Our proteomic survey focused to a set of PTMs described as advanced glycation-end products (AGEs) or advanced lipooxidation-end products (ALEs), known to accumulate during aging and age-associated diseases. AGEs and ALEs are the result of non-enzymatic reactions of protein with reducing sugars, or with oxidized sugar or lipid degradation products, which may alter and sometimes crosslink the positively charged amino acids lysine and arginine on proteins. Our proteomic dataset supported detection of several AGEs and ALEs, including the adducts 4-ONE (4-oxononenal), 4-ONE+Delta:H(-2)O(-1) (dehydrated 4-oxononenal Michael adduct), carboxyethyl, carboxymethyl, 3-deoxyglucosone derived dihydroxyimidazoline, G-H1 (glyoxal-derived hydroimidazolone), HNE (4-hydroxynonenal), several carbonylated adducts including proline to pyrrolidinone or pyrrolidone Other detected modifications of note include mono-oxidation and di-oxidation products, including tryptophan to kynurenine. We also detected lysine-epsilon-gly-gly (GlyGly), which marks ubiquitylated sites, indicating potential substrates of proteasome-dependent degradation. The grand total PTM change was negative for treated aged OA mice but positive for treated young OA mice, regardless of whether summed across modifications or across proteins. In other words, senolytic treatment reduced oxidation associated PTMs more effectively in aged OA mice than in young OA mice. The LFQ proteomics analysis was complemented with new biophysical computational tools aimed to predict the stability of carbonylated proteins extracted from the joints. The biophysical model predicted divergent proteomic responses between young and aged animals. Altogether, our results showed that senolysis reduces overall oxidative stress in the aged arthritic joints in vivo.

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Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase

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