IndraLab
Statements
sparser
"Of note, the BAP1-UCH loop is larger than the ones in other UCH proteins, with a high conservation of both these loop amino acids and of multiple amino acids structurally near this loop ( xref , xref , xref ), implying that larger substrates may be available to BAP1’s catalytic cleavage site than for other UCH domains, yielding a BAP1-specific recruitment of proteins/domains such as ASX and ULD for enzyme regulation ( xref )."
sparser
"In contrast to primary nucleation, secondary nucleation is influenced by local environmental fluctuations and molecular noise, which can lead to variable onset times and heterogeneous spatial distribution of aggregates in cells, as reflected in the variability observed in global fitting of k 2 and n 2 parameters for BAP1‐UCH variants (Table xref and S2, Supporting Information)."
sparser
"The plasmid encoding WT BAP1‐UCH and its variants N78S, C91W, F81V, and G128R, available in our lab, were used to over‐express and purify the proteins using the same protocol as published before. [ xref ] Briefly, the purification steps include affinity chromatography with Ni‐NTA resin, cleavage of the histidine tag with a 1:50 ratio of Tobacco Etch Virus (TEV) protease and protein of interest containing tag, and size‐exclusion chromatography."
sparser
"Using computer molecular modeling of UCHL5 structures, we predicted that the BAP1-ULD domain folds back to the BAP1-UCH catalytic domain and that the ASXL2-AB box stabilizes the UCH catalytic loop via a unique BAP1 mechanism not seen in other UCH proteins, allowing for ubiquitin to fit into the active site ( xref , xref )."
sparser
"We have demonstrated that ASXL, via its AB box, acts as a molecular scaffold to recruit the BAP1-ULD domain to transcription factors that bind to specific target genes; the BAP1-UCH catalytic domain then removes ubiquitin from histones on chromatin to regulate the expression of these transcriptional targets ( xref )."
sparser
"To investigate the mechanism by which BAP1‐UCH variant aggregates, we systematically analyze the concentration‐dependent aggregation kinetics (Figure xref and Figure S1 and S2, Supporting Information), secondary structure transitions (Figure xref ), oligomerization rates (Figure xref ), and morphological features (Figure xref ) of the fibril structures of all variants."
sparser
"Our results reveal that all BAP1‐UCH variants follow a secondary nucleation‐dominated aggregation model, exhibiting strong concentration dependence and significantly higher aggregation rates, which may be responsible for their impaired nuclear import, leading to increased cytosolic retention."
sparser
"Analysis of the COSMIC (Catalogue of Somatic Mutations in Cancer) database reveals that over half of the documented cancer‐associated mutations reside within the BAP1‐UCH domain. [ xref ] Hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) analyses have revealed enhanced structural perturbations among several cancer‐associated BAP1‐UCH variants (S63C, N78S, F81V, C91W, and G128R) compared with the WT. [ xref ] Except for S63C, these variants display markedly increased ThT fluorescence intensity, suggesting their potential for amyloid formation. [ xref ] Immunofluorescence microscopy observations indicate decreased nuclear localization in the pathological and amyloidogenic BAP1‐UCH variants (I47F, F81V, A95D, and G178V). [ xref ] "
sparser
"Cancer‐associated mutations within the ubiquitin C‐terminal hydrolase (UCH) domain of BAP1 (BAP1‐UCH) are implicated in direct and indirect functional loss: catalytic mutations, namely C91G, C91W, H169Q directly contribute to loss of the DUB activity; the S10N mutation perturbs ubiquitin binding thereby leading to functional loss; in contrast, N78S, F81V, and G128R significantly destabilize BAP1‐UCH that lead to aggregation and indirectly contribute to the functional loss."
sparser
"F81V and G128R exhibited faster aggregation kinetics relative to those of wild type (WT), N78S, and C91W. The protein concentration‐dependent ThT kinetic traces were fit to different amyloid formation models by AmyloFit, [ xref ] and a secondary nucleation‐dominated model was found to best describe the aggregation mechanism of these BAP1‐UCH variants."
sparser
"In contrast, N78S and C91W exhibited prominent aggregation behaviors at moderate protein concentrations, while F81V and G128R were the most aggregation‐prone: G128R was readily aggregated at 1 µM. By plotting the half‐times of the individual variants as a function of protein concentration, we could further separate the aggregation behaviors of the BAP1‐UCH variants into two groups: F81V and G128R exhibited shallow slopes in the double logarithm plots, whereas N78S, C91W, and WT exhibited steeper slopes, indicating that the aggregation propensities of F81V and G128R are less sensitive to concentration fluctuations relative to those of N78S, C91W, and WT (Figure xref )."
sparser
"Furthermore, the plateau values of BAP1‐UCH variants were not always the same as that of WT, particularly for C91W and G128R. This may be attributed to the differences in the amyloid structures that induce different intercalated ThT binding modes in the β ‐sheets. [ xref ] There may be some issues with the very high aggregation propensity of G128R that caused some precipitation during the ThT kinetic measurements, which resulted in the loss of ThT fluorescence."
sparser
"To further assess the oligomeric states of BAP1‐UCH variants, we employed size‐exclusion chromatography‐coupled multiangle light scattering (SEC‐MALS) to estimate the oligomeric state distributions and the corresponding molecular weights (MWs) (Figure S5, Supporting Information)."
sparser
"WT was the only exception that the population of peak 1 accounted for only ≈2% of the overall population, whereas the remaining population corresponded to the slowest elution peak (peak 3) has an estimated MW (≈30 kDa) closely matching the expected MW of a monomeric BAP1‐UCH (Figure S5A–C, Supporting Information)."
sparser
"When normalized with respect to the UV absorption of peak 3, the intrinsic FL of peak 3 was lower than that of peak 2 in all BAP1‐UCH mutants, suggesting that the higher order structure of BAP1‐UCH variants may be partially unfolded, leading to FL quenching (Figure S5A, Supporting Information), reminiscent of our previous observation for urea‐unfolded UCH‐L1, a paralog of BAP1‐UCH. [ xref ] "
sparser
"Collectively, our SEC‐MALS analyses of the BAP1‐UCH variants at their early onset of the aggregation processes showed that the aggregation involves the formation of relatively small oligomers (dimers and trimers for F81V and G128R, respectively, and tetramer to pentamer for N78S) before quickly aggregating into very large soluble aggregates between 20 MDa and 300 MDa."
sparser
"To test whether the experimental aggregation propensities of individual BAP1‐UCH variants can be predicted reliably by computational tools, we used AGGRESCAN to estimate the aggregation propensity as a function of protein sequence. [ xref ] The results indicated that N78S and C91W exhibit higher aggregation scores in the regions near the mutation sites compared to WT (Figure S7, Supporting Information)."
sparser
"Contrary to the experimental observations, AGGRESCAN predicted lower aggregation scores for F81V and G128R. We next turned to a structure‐based aggregation propensity predictor, AGGRESCAN4D, to identify aggregation‐prone regions based on the structural model of BAP1‐UCH predicted by AlphaFold. [ xref , xref – xref ] The predicted aggregation propensities show the following ranking order: F81V > N78S, C91W > G128R > WT (Table S2, Supporting Information)."
sparser
"Homology of the BAP1-UCH and other UCH-like proteins infers that this BAP1 motif functions in either ubiquitin-mediated, proteasomal degradation or some other ubiquitin-facilitated regulatory pathways that are involved in BRCA1 function, cellular proliferation, differentiation, and/or homeostatic processes ( xref , xref , xref )."
sparser
"On the other hand, the mutation sites of N78S, F81V, and G128R are distant from the catalytic site, which allosterically alter the structures and folding stability of BAP1‐UCH. [ xref ] The degrees of destabilization induced by different cancer‐associated mutations cannot be reliably recapitulated by theoretical sequence‐ and structure‐based predictors of aggregation propensity and folding stability."
sparser
"ThT fluorescence analyses show that all BAP1‐UCH variants exhibit concentration‐dependent aggregation kinetics, and concentration dependency is higher for WT, N78S, and C91W; it is lower for F81V and G128R, likely due to their substantially decreased folding stabilities. [ xref ] AmyloFit analyses indicate that the aggregation of all BAP1‐UCH variants studied herein follows the secondary nucleation‐dominated mechanism (Figure xref ), which is often found in intrinsically disordered proteins (IDPs)."