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Proteasome conformational states during FAST assembly. (A) Sequence of b subunit propeptide regions in WT and FAST mutants. Residues in red are the mutated E (glutamate) and D (aspartate) to A (alanine). (B) Bar graph of percentage of the simulated time frame in DÀ and Dþ states for both WT and FAST b subunit mutants HPs. Error bars signify mean 5 SE for 3 MD simulations of 2.5 us. (C) Bar graph showing the total number of hydrogen bonds formed between the propeptide and the key residues as an average over 3 MD simulations for both WT and FAST HPs. Error bars show mean 5 SE for 3 MD simulations of 2.5 us. (D) 4-20% Trisglycine native gels from in vitro assembly assays at increasing time points (time points labeled above each lane in minutes) for WT and FAST b subunit mutants. Gels were stained with Spyro Ruby protein and visualized with a BioRad Imager. To see this figure in color, go online.
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The 20S proteasome Core Particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form, but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits,...
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... fs, and the coordinates were saved every 0.24 ns. All systems were simulated under periodic boundary conditions in the NPT ensemble. Additional details of the equilibration and MD simulations are provided in section S10 of the supporting material. All simulations for the WT and mutants had a backbone root mean-square deviation around 3.5-6.5 A ˚ (Fig. S4). We found that, after around 500 ns of simulation, the system energy approached a constant average and did not systematically change over the remainder of the simulations (Fig. S3). As a result, we did not include the first 500 ns of the runs in our quantitative ...
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... with our hypothesis, simulation results revealed that the SLOW mutant propeptide resides in the DÀ much more frequently than the WT (Fig. 3 C). We found that after 500 ns, the energy stabilized around a constant average in our simulations, so frames from the first 500 ns are ignored in this analysis (Figs. S3 and S4). In particular, the WT HP exists in the Dþ state about 25% of the time across the three simulated replicates and in the DÀ state 75% of the time (Fig. 3 C). In contrast, the HP of the SLOW mutant nearly always (>99% of simulated time) resides in a DÀ state (Fig. 3 C). To further characterize the difference between the two mutants, we ...
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... that reduce the level of interactions between the propeptide and the key residues should favor the Dþ state and thus lead to faster dimerization. To test this idea directly, we computationally mutated two charged residues in WT region III, glutamate E-9 and aspartate D-12, to alanine using CHARMM GUI (29) to generate what we term the FAST mutant (Fig. 4 A). In the WT HP simulations, both residues interact with the key residues, specifically by forming side-chain hydrogen bonds with several amino acids that form salt bridges across the HP dimerization interface, particularly R29 and N24 of the b subunit. We thus hypothesized that mutating these residues to alanine could potentially ...
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... of these simulations revealed that interactions between the propeptide and the key dimerization residues were less frequent compared with WT, with the FAST mutant spending close to 50% of the simulation in the Dþ state (Fig. 4 B). Furthermore, the FAST HP formed fewer hydrogen bonds with key residues compared with the WT HP (Fig. 4 C). As expected, interactions between the À9 and À12 residues of the propeptide and the key residues were much less frequent in FAST than WT (Fig. S7). This suggested that the FAST mutant would indeed dimerize faster than ...
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... of these simulations revealed that interactions between the propeptide and the key dimerization residues were less frequent compared with WT, with the FAST mutant spending close to 50% of the simulation in the Dþ state (Fig. 4 B). Furthermore, the FAST HP formed fewer hydrogen bonds with key residues compared with the WT HP (Fig. 4 C). As expected, interactions between the À9 and À12 residues of the propeptide and the key residues were much less frequent in FAST than WT (Fig. S7). This suggested that the FAST mutant would indeed dimerize faster than ...
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... test our predictions experimentally, we generated a double point mutation D-12A/E-9A variant (Fig. 4 ...
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... compare the assembly rates of WT and FAST mutants, a and b mutant substrates were mixed in vitro and incubated at 30 C at increasing time points from 0 to 180 min, and we performed native PAGE on the reaction products (3,8,22). This assay provides a readout of assembly kinetics of both the WT and FAST mutant HP and CP (Fig. 4 D). Consistent with previous experimental results (4,13,7), we observed that WT HP formation is essentially complete at our earliest time point (0 min), which is obtained by mixing the subunits, immediately loading the reaction on the gel, and then running the gel. In the WT case, CP starts to appear after 15 min and continues to increase ...
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... we observed that WT HP formation is essentially complete at our earliest time point (0 min), which is obtained by mixing the subunits, immediately loading the reaction on the gel, and then running the gel. In the WT case, CP starts to appear after 15 min and continues to increase in concentration as indicated by the increase in band intensity (Fig. 4 D). In contrast, the FAST mutant shows significantly more CP formation at the 0 and 15 min time points, with CP assembly essentially complete by 30 min. These findings demonstrate that the FAST mutant does indeed dimerize considerably more rapidly than the ...
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... that we expected would reduce the number of interactions between the propeptide and the key residues, thus shifting the equilibrium to the Dþ state and increasing the dimerization rate. Specifically, we chose two negatively charged residues in the propeptide that frequently interacted with the key residues in our WT simulations: E-9 and D-12 (Fig. 4 A). Next, we computationally mutated these residues to alanine to generate what we call the FAST mutant, and, as expected, we saw a shift in the equilibrium that favored the Dþ state and a corresponding decrease in the total number of interactions between the key residues and the propeptide in our MD simulations (Fig. 4 B and C). We then ...
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... simulations: E-9 and D-12 (Fig. 4 A). Next, we computationally mutated these residues to alanine to generate what we call the FAST mutant, and, as expected, we saw a shift in the equilibrium that favored the Dþ state and a corresponding decrease in the total number of interactions between the key residues and the propeptide in our MD simulations (Fig. 4 B and C). We then made this mutant in the lab and tested its assembly kinetics experimentally and found that it did indeed dimerize more quickly than WT (Fig. 4 D). This finding is consistent with our hypothesis and suggests that the frequency of interactions between the propeptide and the key residues likely plays an essential role in ...
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... saw a shift in the equilibrium that favored the Dþ state and a corresponding decrease in the total number of interactions between the key residues and the propeptide in our MD simulations (Fig. 4 B and C). We then made this mutant in the lab and tested its assembly kinetics experimentally and found that it did indeed dimerize more quickly than WT (Fig. 4 D). This finding is consistent with our hypothesis and suggests that the frequency of interactions between the propeptide and the key residues likely plays an essential role in regulating the dimerization ...
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... prevented. The FAST mutant, however, forms fewer interactions, and as a result, the transitions out of the DÀ state are much faster than WT (Fig. 6 C). As a result, the FAST mutant more frequently visits states where all the key residues are available for dimerization, and this likely allows dimerization kinetics to proceed far more rapidly (Fig. 4 D). Simple models of the dimerization process indicate that the Dþ/DÀ conformational transition can indeed explain both the separation of timescales in HP dimerization and the differences between the mutants, even when accounting for the finite lifetime of the HP-HP encounter complex (supporting information section S12) ...
Citations
... The function of the proteasome affects patient survival and glioma cell proliferation and invasion, but the mechanism of its action remains unclear 8 . The 20S proteasome consists of four stacked rings each with seven subunits formed by 14 distinct gene products into a four-layered structure 9 . Seven subunits of α and β core particles are encoded by PSMA1-7 10 and PSMB1-7 11 . ...
There has been an upward trend in the incidence of glioma, with high recurrence and high mortality. The beta subunits of the 20S proteasome are encoded by the proteasome beta (PSMB) genes and may affect the proteasome’s function in glioma, assembly and inhibitor binding. This study attempted to reveal the function of the proliferation and invasion of glioma cells, which is affected by proteasome 20S subunit beta 2 (PSMB2). We subjected the data downloaded from the TCGA database to ROC, survival, and enrichment analyses. After establishing the stable PSMB2 knockdown glioma cell line. We detect the changes in the proliferation, invasion and migration of glioma cells by plate colony formation assay, transwell assay, wound healing assay and flow cytometry and PSMB2 expression was verified by quantitative PCR and Western blotting to identify the mRNA and protein levels. PSMB2 expression was higher in glioma tissues, and its expression positively correlated with poor prognosis and high tumor grade and after PSMB2 knockdown, the proliferation, invasion and migration of glioma cells were weakened.
... M.tb proteasomes have been purified as an assembled complex by co-expressing the 4 4 vitro. This is in contrast to the CP from the related actinomycete bacterium Rhodococcus erythropolis (R.e), where these two proteins can be readily expressed separately as monomers (8) (9)(10)(11). Negative stain electron microscopy (EM) images of M.tb proteasomes show a conserved four stacked ring structure similar to the 20S proteasome from R.e, the archaeon Thermoplasma acidophilum, yeast and humans (2). ...
... Assembly time courses resolved on native gels revealed clear HP and CP bands, similar to those observed with the R.e subunits. In R.e, there is a distinct separation of time scales between HP formation (which happens extremely rapidly) and HP dimerization, which under standard experimental conditions can take one or two hours to proceed to completion (11,12). We found a similar separation of timescales in the M.tb proteasome, but under similar conditions dimerization was much slower, only going to completion over the course of 12-24 hours. ...
... Taken together, these findings strongly indicate that the assembly pathway is conserved between M.tb and R.e and likely all bacteria. Both self-assembly experiments and MD simulations of the HP from R.e suggest that a flexible region of the β subunit (specifically, a part of the β propeptide that is cleaved off during autocatalytic activation of the CP) is responsible for regulating the timescale of HP dimerization (8,11). It is unclear if changes to the sequence of this region in M.tb are responsible for the dramatic decrease in the rate of HP dimerization in this case. ...
According to the WHO, one in three people in the world has a latent tuberculosis infection. Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (M.tb). The development of multi-drug resistant (MDR) tuberculosis indicates a need for novel treatments. Hence, it is important to find a second line of treatment for patients infected with MDR tuberculosis. The proteasome is known to be necessary for survival under stress and pathogenicity in M.tb. However, our ability to use the proteasome as drug target has been limited by our abilities to screen for inhibitor compounds in vitro. The proteasome is a protease complex that degrades proteins and is crucial for the maintenance of protein homeostasis within cells. Like many protein complexes, the proteasome must assemble into a specific quaternary structure in order to be active. Specifically, the proteolytically-active proteasome Core Particle (CP) consists of 28 subunits (14 α and 14 β) that must assemble into a barrel-like structure in order to become catalytically active. Hence, understanding the assembly process in not only important from a basic cell biological perspective, but may also serve as the basis for the discovery of novel assembly inhibitors. In this study, we have established for the first time a protocol to express and purify the M.tb α and β subunits separately in vitro. The subunits are soluble monomers on purification and only assemble into active CPs upon reconstitution. Our assembly experiments revealed that M.tb CP assembly pathway is almost certainly identical to that seen in previous experiments on the CP from the bacterium Rhodococcus erythropolis (R.e), but assembly in M.tb is much slower. Interestingly, we found that subunits from M.tb and R.e spontaneously self-assembled into active hybrid proteasomes on reconstitution with each other, despite having only 65% sequence similarity. Our work thus strongly suggests that the CP assembly pathway is conserved across bacteria, and the ability to perform in vitro assembly experiments on the M.tb proteasome opens up the possibility of performing critical experiments, including screening for potential molecules that could inhibit assembly, directly in this clinically-relevant organism.
... The function of the proteasome affects patient survival and glioma cell proliferation and invasion, but the mechanism of its action remains unclear [8] . The 20S proteasome consists of four stacked rings each with seven subunits formed by 14 distinct gene products into a four-layered structure [9] . Seven subunits of α and β core particles are encoded by PSMA1-7 [10] and PSMB1-7 [11] . ...
There has been an upward trend in the incidence of glioma, with high recurrence and high mortality. The beta subunits of the 20S proteasome are encoded by the proteasome beta (PSMB) genes and may affect the proteasome's function in glioma, assembly and inhibitor binding. This study attempted to reveal the function of the proliferation and invasion of glioma cells, which is affected by proteasome 20S subunit beta 2 (PSMB2). We subjected the data downloaded from the TCGA database to ROC, survival, and enrichment analyses. PSMB2 expression was verified by quantitative PCR and Western blotting to identify the mRNA and protein levels. PSMB2 expression was higher in glioma tissues, and its expression positively correlated with poor prognosis and high tumor grade.
There has been an upward trend in the incidence of glioma, with high recurrence and high mortality. The beta subunits of the 20S proteasome are encoded by the proteasome beta (PSMB) genes and may affect the proteasome's function in glioma, assembly and inhibitor binding. This study attempted to reveal the function of the proliferation and invasion of glioma cells, which is affectedby proteasome 20S subunit beta 2 (PSMB2). We subjected the data downloaded from the TCGA database to ROC, survival, and enrichment analyses. PSMB2 expression was verified by quantitative PCR and Western blotting to identify themRNA and protein levels. PSMB2expressionwas higher in glioma tissues, and its expression positively correlated with poor prognosis and high tumor grade.
