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• W3134459642 abstract "•Ub half-life in mammalian cells is 4 h•On average six Ub molecules are conjugated with a substrate•One Ub moiety is lost per four episodes of engagement of substrate by proteasome•Single lysine residue K27 is critical for the stability of the whole Ub molecule Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art. Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art. The ubiquitin-proteasome system (UPS), consisting of hundreds of members, is involved in critical intracellular processes such as differentiation, DNA repair, apoptosis, autophagy, cell cycle progression, protein quality control, regulation of transcription, and generation of major histocompatibility complex (MHC) class I-associated peptides. The absolute majority of proteins require conjugation with several ubiquitin (Ub) molecules prior to proteasome-mediated degradation (Ciechanover and Stanhill, 2014Ciechanover A. Stanhill A. The complexity of recognition of ubiquitinated substrates by the 26S proteasome.Biochim. Biophys. Acta. 2014; 1843: 86-96Crossref PubMed Scopus (111) Google Scholar). Typically, ε-amino group of an internal lysine residue of the substrate forms an isopeptide bond with a carboxyl-terminal Gly76 of the first Ub. Further growing of polyubiquitin (polyUb) chains is accomplished by conjugation of the coming Ub molecule to Lys48 of the previously added Ub (Chau et al., 1989Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1108) Google Scholar; Hershko and Ciechanover, 1998Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6825) Google Scholar). The initial discovery of the ATP-dependent ubiquitination system (Ciechanover et al., 1981Ciechanover A. Heller H. Katz-Etzion R. Hershko A. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.Proc. Natl. Acad. Sci. U S A. 1981; 78: 761-765Crossref PubMed Scopus (158) Google Scholar) was consistently complemented with further observation of a 2.5-MDa multicatalytic proteinase complex called the “proteasome” (Arrigo et al., 1988Arrigo A.P. Tanaka K. Goldberg A.L. Welch W.J. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome).Nature. 1988; 331: 192-194Crossref PubMed Scopus (316) Google Scholar). Further studies revealed that the 26S proteasome contains a 19S regulatory sub-particle (Lander et al., 2012Lander G.C. Estrin E. Matyskiela M.E. Bashore C. Nogales E. Martin A. Complete subunit architecture of the proteasome regulatory particle.Nature. 2012; 482: 186-191Crossref PubMed Scopus (470) Google Scholar) capping a 20S proteolytic core particle that is responsible for recognition (Deveraux et al., 1994Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates.J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar), unfolding (Benaroudj et al., 2003Benaroudj N. Zwickl P. Seemuller E. Baumeister W. Goldberg A.L. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation.Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar; Smith et al., 2005Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins.Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), deubiquitination (Verma et al., 2002Verma R. Aravind L. Oania R. McDonald W.H. Yates 3rd, J.R. Koonin E.V. Deshaies R.J. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.Science. 2002; 298: 611-615Crossref PubMed Scopus (830) Google Scholar), and final translocation of the substrate into the antechamber (de la Pena et al., 2018de la Pena A.H. Goodall E.A. Gates S.N. Lander G.C. Martin A. Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.Science. 2018; 362: eaav0725Crossref PubMed Scopus (165) Google Scholar; Ruschak et al., 2010Ruschak A.M. Religa T.L. Breuer S. Witt S. Kay L.E. The proteasome antechamber maintains substrates in an unfolded state.Nature. 2010; 467: 868-871Crossref PubMed Scopus (100) Google Scholar). The molecular mechanism of engagement of classic K48-polyubiquitinated substrate with 26S proteasome was recently resolved on the structural level (Lander et al., 2012Lander G.C. Estrin E. Matyskiela M.E. Bashore C. Nogales E. Martin A. Complete subunit architecture of the proteasome regulatory particle.Nature. 2012; 482: 186-191Crossref PubMed Scopus (470) Google Scholar; Matyskiela et al., 2013Matyskiela M.E. Lander G.C. Martin A. Conformational switching of the 26S proteasome enables substrate degradation.Nat. Struct. Mol. Biol. 2013; 20: 781-788Crossref PubMed Scopus (182) Google Scholar); herewith, numerous alternative combinations of Ub chains are readily recognized by the proteasome (Kravtsova-Ivantsiv and Ciechanover, 2012Kravtsova-Ivantsiv Y. Ciechanover A. Non-canonical ubiquitin-based signals for proteasomal degradation.J. Cell Sci. 2012; 125: 539-548Crossref PubMed Scopus (159) Google Scholar). Even more complicated aspects of proteasome-substrate interaction were uncovered with the discovery of “Ub-like” UBA-UBL shuttle proteins (Hiyama et al., 1999Hiyama H. Yokoi M. Masutani C. Sugasawa K. Maekawa T. Tanaka K. Hoeijmakers J.H. Hanaoka F. Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.J. Biol. Chem. 1999; 274: 28019-28025Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar; Wilkinson et al., 2001Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains.Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (347) Google Scholar). At present, it is suggested that the signal for proteasome-mediated degradation consists of two essential elements, namely Ub conjugated to the polypeptide chain or any alternative species able to fix the substrate on the proteasome and intrinsic unfolded fragment in the substrate (Prakash et al., 2004Prakash S. Tian L. Ratliff K.S. Lehotzky R.E. Matouschek A. An unstructured initiation site is required for efficient proteasome-mediated degradation.Nat. Struct. Mol. Biol. 2004; 11: 830-837Crossref PubMed Scopus (356) Google Scholar). The last serves as a translocation initiation site. It promotes entry of the substrate into the ATPase ring, which induces its rearranging accomplished by ATP hydrolysis to translocation-competent conformation with a widened coaxially aligned central channel (Matyskiela et al., 2013Matyskiela M.E. Lander G.C. Martin A. Conformational switching of the 26S proteasome enables substrate degradation.Nat. Struct. Mol. Biol. 2013; 20: 781-788Crossref PubMed Scopus (182) Google Scholar). Despite intensive studying of Ub, at the present moment most of the data are based on the in vitro experiments; therefore, peculiarities of its metabolism are still poorly resolved. The complexity of the study of Ub turnover is explained by its unique structure that restricts fusion with fluorescent proteins and tags longer than 20 amino acids, difficulties with immunoprecipitation of polyubiquitin conjugates, and the impossibility of quantitative analysis by blotting techniques. Here we accurately determined fundamental parameters of Ub metabolism utilizing its real-time fluorescent tracking. Here we claim the PRIME (probe incorporation mediated by enzymes) technique based on mutated Escherichia coli lipoic acid ligase (LplA) that conjugates the ε-amino group of lysine in a short 13-amino acid peptide tag with chemical fluorophores (Cohen et al., 2011Cohen J.D. Thompson S. Ting A.Y. Structure-guided engineering of a Pacific Blue fluorophore ligase for specific protein imaging in living cells.Biochemistry. 2011; 50: 8221-8225Crossref PubMed Scopus (34) Google Scholar; Uttamapinant et al., 2010Uttamapinant C. White K.A. Baruah H. Thompson S. Fernandez-Suarez M. Puthenveetil S. Ting A.Y. A fluorophore ligase for site-specific protein labeling inside living cells.Proc. Natl. Acad. Sci. U S A. 2010; 107: 10914-10919Crossref PubMed Scopus (243) Google Scholar) as a methodology for real-time and endpoint monitoring of Ub metabolism in physiological conditions. In the present study, we used triple mutant E20A/F147A/H149G LplA ligase (herein after referred as LplA(AAG)) for intracellular labeling of recombinant proteins containing ligase acceptor peptide (LAP) with a derivative of the low-molecular-weight fluorophore resorufin, emitting at 595 nm (Liu et al., 2014Liu D.S. Nivon L.G. Richter F. Goldman P.J. Deerinck T.J. Yao J.Z. Richardson D. Phipps W.S. Ye A.Z. Ellisman M.H. et al.Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.Proc. Natl. Acad. Sci. U S A. 2014; 111: E4551-E4559Crossref PubMed Scopus (55) Google Scholar). We adjusted the previously described scheme of synthesis of a cell-penetrating resorufin derivative (please refer to STAR methods) with acetoxymethyl-blocked acidic groups (resorufin-AM2). The most crucial alteration was introduced in the last stage, where Hünig base (N,N-diisopropylethylamine) was used instead of triethylamine. This modification resulted in an increase of the yield of the trans to the cis product, which was observed in the ratio 5:1 (Figures S1A), compared with 1:10 as reported previously (Liu et al., 2014Liu D.S. Nivon L.G. Richter F. Goldman P.J. Deerinck T.J. Yao J.Z. Richardson D. Phipps W.S. Ye A.Z. Ellisman M.H. et al.Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.Proc. Natl. Acad. Sci. U S A. 2014; 111: E4551-E4559Crossref PubMed Scopus (55) Google Scholar). To synchronize expression of LplA(AAG) and the protein under investigation, we created lentiviral vectors containing cDNA coding for the protein of interest in frame with a 3FLAG tag and LAP and fusion protein ZsGreen-LplA(AAG) connected by an internal ribosome entry site (IRES) (Figure 1). Stably transduced cells expressing proteasome-substrate myelin basic protein (Belogurov et al., 2014Belogurov Jr., A. Kudriaeva A. Kuzina E. Smirnov I. Bobik T. Ponomarenko N. Kravtsova-Ivantsiv Y. Ciechanover A. Gabibov A. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.J. Biol. Chem. 2014; 289: 17758-17766Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) (MBP) fused with LAP (MBP-LAP) and ZsGreen-LplA(AAG) were pulsed (i.e., incubated with resorufin-AM2, and further intensively washed) for 20 and 60 min with various concentrations of resorufin-AM2. Stained cells were lysed and subjected to the polyacrylamide gel electrophoresis (PAGE) with subsequent double-color fluorescent imaging. A resorufin-AM2 concentration of 5 μM and 20-min pulse with fluorophore were chosen as the optimal conditions for the intracellular labeling (Figure S1B). We next tested PRIME in a “pulse-chase” mode and accurately measured the half-lives (τ1/2) of MBP (Figures 2A, S1C and S1D), and the stable protein dihydrofolate reductase (DHFR) (Figures 2B and S1E), using either PRIME or cycloheximide (CHX) chase. To this end, we transduced HEK293T cells with lentivirus coding for the ZsGreen-LplA(AAG) together with either MBP-LAP or DHFR-LAP. Cells were further pulsed with resorufin-AM2, incubated for the indicated time points, and subjected the cell lysates to the PAGE with subsequent double-color fluorescent imaging and western blotting. Resorufin-labeled MBP was utterly degraded in 6 h regardless of CHX addition, whereas the amount of total MBP, measured by western blotting, was decreased only on CHX administration. The τ1/2 of intracellular MBP measured by the PRIME approach in the presence or absence of CHX was 1.4 and 1.8 h, respectively (Figure 2C). Next, we estimated the τ1/2 of DHFR using PRIME-based methodology. The observed τ1/2 = 9 h (Figure 2C) was in a good agreement with previously reported values of DHFR τ1/2 measured in a 35S methionine radiolabeled pulse-chase experiment (Hsieh et al., 2013Hsieh Y.C. Tedeschi P. Adebisi Lawal R. Banerjee D. Scotto K. Kerrigan J.E. Lee K.C. Johnson-Farley N. Bertino J.R. Abali E.E. Enhanced degradation of dihydrofolate reductase through inhibition of NAD kinase by nicotinamide analogs.Mol. Pharmacol. 2013; 83: 339-353Crossref PubMed Scopus (22) Google Scholar). Routine CHX chase analysis resulted in the determination of a DHFR τ1/2 of about 12.5 h. We additionally compared degradation rate of the nuclear factor of kappa light polypeptide gene enhancer in B cell inhibitor alpha (IkBa) (Figure 2D) and DNA-binding protein inhibitor Id-1 (Figure 2E) utilizing either CHX or resorufin chase. Our data suggest that estimated τ1/2 of both LAP- and FLAG-tagged substrates is in a good agreement with previously reported data (Pennington et al., 2001Pennington K.N. Taylor J.A. Bren G.D. Paya C.V. IkappaB kinase-dependent chronic activation of NF-kappaB is necessary for p21(WAF1/Cip1) inhibition of differentiation-induced apoptosis of monocytes.Mol. Cell Biol. 2001; 21: 1930-1941Crossref PubMed Scopus (69) Google Scholar; Sun et al., 2008Sun L. Trausch-Azar J.S. Muglia L.J. Schwartz A.L. Glucocorticoids differentially regulate degradation of MyoD and Id1 by N-terminal ubiquitination to promote muscle protein catabolism.Proc. Natl. Acad. Sci. U S A. 2008; 105: 3339-3344Crossref PubMed Scopus (44) Google Scholar). Summarizing, in comparison with CHX chase, the PRIME-based technique provides more accurate values for the stable proteins. It seems that the analysis of proteins with τ1/2 of tens of minutes is a challenge for the PRIME-based method because incubation of cells with resorufin-AM2 and consequent washing of cells require at least 20 min.Figure 2Monitoring of protein metabolism using PRIME combined with in-gel fluorescence, fluorescent microscopy, and flow cytometryShow full caption(A and B) HEK293T cells expressing ZsGreen-LplA(AAG) together with either FLAG-tagged MBP-LAP or DHFR-LAP were pulsed with resorufin-AM2 and incubated in the presence or absence of CHX. Cell lysates were analyzed using in-gel fluorescence and western blotting using a mixture of anti-FLAG and anti-actin antibodies. The panels show the overlay of red fluorescence with chemiluminescent western blotting signal (pseudo-blue).(C) The percentage of remaining MBP (top) and DHFR (bottom) in the presence (circles) or absence (squares) of CHX was calculated as the ratio of protein level at the time points indicated relative to the initial protein level, according to the resorufin fluorescence normalized to the ZsGreen fluorescence (red curves) or a-FLAG WB signal normalized to the actin (black curves). τ1/2 are indicated.(D and E) HEK293T cells expressing FLAG- or LAP-tagged IkBα or Id-1 were pulsed with resorufin-AM2 and incubated in the presence (FLAG) or absence (LAP) of CHX. Cell lysates were analyzed according to (B). The percentage of remaining FLAG- (circles) or LAP-tagged (squares) IkBα (D) and Id-1 (E) in the presence or absence CHX was calculated according to (C).(F) HEK293T cells expressing ZsGreen-LplA(AAG) together with either MBP-LAP or DHFR-LAP were pulsed with resorufin-AM2, washed, incubated for indicated time points, and further analyzed by fluorescent microscopy.(G) HEK293T cells co-expressing MBP-LAP and ZsGreen-LplA(AAG) were pulsed with resorufin-AM2 and incubated in the presence or absence of proteasome inhibitor PS-341 (i) and subjected to flow cytometry analysis at indicated time points (ii).(H) Plots represent an overlay of flow cytometry data in dot-plot (i) and histogram (ii) format.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) HEK293T cells expressing ZsGreen-LplA(AAG) together with either FLAG-tagged MBP-LAP or DHFR-LAP were pulsed with resorufin-AM2 and incubated in the presence or absence of CHX. Cell lysates were analyzed using in-gel fluorescence and western blotting using a mixture of anti-FLAG and anti-actin antibodies. The panels show the overlay of red fluorescence with chemiluminescent western blotting signal (pseudo-blue). (C) The percentage of remaining MBP (top) and DHFR (bottom) in the presence (circles) or absence (squares) of CHX was calculated as the ratio of protein level at the time points indicated relative to the initial protein level, according to the resorufin fluorescence normalized to the ZsGreen fluorescence (red curves) or a-FLAG WB signal normalized to the actin (black curves). τ1/2 are indicated. (D and E) HEK293T cells expressing FLAG- or LAP-tagged IkBα or Id-1 were pulsed with resorufin-AM2 and incubated in the presence (FLAG) or absence (LAP) of CHX. Cell lysates were analyzed according to (B). The percentage of remaining FLAG- (circles) or LAP-tagged (squares) IkBα (D) and Id-1 (E) in the presence or absence CHX was calculated according to (C). (F) HEK293T cells expressing ZsGreen-LplA(AAG) together with either MBP-LAP or DHFR-LAP were pulsed with resorufin-AM2, washed, incubated for indicated time points, and further analyzed by fluorescent microscopy. (G) HEK293T cells co-expressing MBP-LAP and ZsGreen-LplA(AAG) were pulsed with resorufin-AM2 and incubated in the presence or absence of proteasome inhibitor PS-341 (i) and subjected to flow cytometry analysis at indicated time points (ii). (H) Plots represent an overlay of flow cytometry data in dot-plot (i) and histogram (ii) format. See also Figure S1. The monitoring of protein metabolism on a single-cell level was recently shown to be important in terms of proteomic studies (Alber et al., 2018Alber A.B. Paquet E.R. Biserni M. Naef F. Suter D.M. Single live cell monitoring of protein turnover reveals intercellular variability and cell-cycle dependence of degradation rates.Mol. Cell. 2018; 71: 1079-1091.e9Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We therefore tested PRIME combined with fluorescent microscopy. The HEK293T cells transduced with lentivirus coding for the ZsGreen-LplA(AAG) together with either MBP-LAP or DHFR-LAP were pulsed with resorufin-AM2 and incubated for indicated time points. Next, intensities of green and red fluorescence were analyzed by fluorescent microscopy (Figure 2F). In line with our previous results, intensity of resorufin fluorescence was rapidly decreased in cells expressing MBP-LAP, in contrast to cells expressing DHFR-LAP. The dynamics of resorufin-pulsed cell decoloration was comparable with timing earlier observed during in-gel fluorescence measurement. This observation suggests that protein-conjugated resorufin upon protein degradation is not significantly retained in the cells and is rapidly washed out. Co-expression of LplA(AAG) and LAP-tagged proteins allowed us to monitor its degradation on a single-cell level. We analyzed changes in the intensity of green and red fluorescence of resorufin-AM2-pulsed HEK293T cells expressing MBP-FLAG-LAP and ZsGreen-LplA(AAG) depending on time utilizing flow cytometry. Resorufin fluorescence in the population of double-positive cells decreased with time and was significantly less changed in cells incubated with medium supplemented with proteasome inhibitor PS-341 (Figure 2G). Plotting of resorufin fluorescence of resorufin-AM2-pulsed HEK293T cells expressing MBP-FLAG-LAP and ZsGreen-LplA(AAG) in comparison with non-transduced cells demonstrated that MBP-expressing cells are entirely overlapped with non-transduced cells after 2 h of incubation with medium (Figure 2H). We next analyzed the metabolism of Ub molecule N-terminally fused with LAP (LAP-Ub) (Figure 3A). Resulting N-terminal extension of Ub was 14 amino acids, therefore this tail in principle should not destabilize it (Shabek et al., 2009Shabek N. Herman-Bachinsky Y. Ciechanover A. Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation.Proc. Natl. Acad. Sci. U S A. 2009; 106: 11907-11912Crossref PubMed Scopus (60) Google Scholar). C terminus was left intact to preserve the ability of LAP-Ub to act as a wild-type (WT) Ub in conjugation machinery (Figure S2A). HEK293T cells stably expressing LAP-Ub and ZsGreen-LplA(AAG) were treated by DMSO, proteasome inhibitor PS-341, and inhibitors of deubiquitination enzymes (DUBs) PR-619 and WP1130 for 6 h and further labeled with resorufin-AM2 (Figure 3Ai). Immediately after washing, cells were lysed and subjected to PAGE. Analysis of in-gel resorufin fluorescence resulted in the detection of classic traces representing polyUb chains conjugated to numerous intracellular proteins (Figure 3Aii). The intensity of these traces significantly enhanced in the case of inhibited proteasome or DUBs. We next exposed HEK293T cells stably expressing LAP-Ub to the PS-341 and further labeled cells with resorufin-AM2. Cell lysates were supplemented with PS-341 and PYR-41 and incubated for 2 h in the presence of the various inhibitors of the DUBs (Figure 3B). Endogenous DUBs existing in the cellular lysate significantly reduce intensity of the trace corresponding to the polyubiquitinated proteins while it was preserved in the samples treated by the DUBs' inhibitors. Alternatively, LAP-Ub-expressing HEK293T cells were firstly pulsed with resorufin-AM2 and further incubated with PS-341 or DMSO for 6 h, followed by fluorescent microscopy. Resorufin fluorescence was clearly detected in cells exposed to PS-341, whereas it was almost completely disappeared in DMSO-treated cells (Figure 3C). Fluorescence microscopy of HEK293T cells stably expressing LAP-Ub and ZsGreen-LplA(AAG) pulsed with resorufin-AM2 and further incubated with a medium in the presence or absence of PS-341 revealed that intracellular resorufin fluorescence in DMSO-treated cells is significantly diminished after 4–5 h (Figure 3D), in contrast to those exposed to the PS-341. To accurately estimate the τ1/2 of Ub in mammalian cells, we pulsed HEK293T cells stably expressing LAP-Ub and ZsGreen-LplA(AAG) with resorufin-AM2. Cells were further incubated with medium with or without PS-341, lysed at indicated time points, followed by measurement of the intensity of polyUb conjugates visualized by in-gel fluorescence (Figures 3Ei, S2B, and S2C). Profiling of Ub traces revealed that their intensity decreased twice in ~4 h (Figures 3Eii and S3), and unlabeled Ub completely replace resorufin-labeled Ub in polyUb chains in 10 h. In the presence of PS-341, total amount of polyUb conjugates increased by up to a quarter during the first 2 h, similar to an observation reported by Choi et al., 2019Choi Y.S. Bollinger S.A. Prada L.F. Scavone F. Yao T. Cohen R.E. High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.Nat. Methods. 2019; 16: 771-777Crossref PubMed Scopus (16) Google Scholar and further gradually decreased (Figure 3Eii). We reasoned that, in the first hours after proteasome inhibition, Ub ligases generally overcome DUBs. Thus, substrates, especially with high molecular weight, which are generally more stable, tend to become “overubiquitinated”. During the next 2–3 h, newly synthesized substrates with rapid turnover, which start to accumulate in the presence of the proteasome inhibitor, are massively ubiquitinated and therefore exhaust the pool of the unconjugated Ub. After 3 h, the pool of free Ub seems to be depleted (Figure 4A) and DUBs should start to remove Ub from the overubiquitinated proteins in order to supply Ub to the ligases. The pool of unconjugated Ub is stabilized after 6 h of exposure to the PS-341, which means a balance is established between Ub ligases and DUBs. To study the distribution of Ub monomers in the polyUb chains, we performed ultrasensitive quantitative scanning of polyUb traces resolved by gradient polyacrylamide gel (PAAG). For this purpose, we analyzed lysates of the HEK293T cells stably expressing LAP-Ub and ZsGreen-LplA(AAG) pulsed with resorufin-AM2 and further incubated with a medium in the presence or absence of PS-341 for 6 h (Figures 4B and S4A). Overlay of the intensity of resorufin fluorescence depending on the total mass of protein-Ub conjugates at the starting point and after 6 h of incubation with PS-341 revealed that, in the case of the proteasome inhibition, resorufin-labeled Ub was redeployed from conjugates with molecular weight more than 170 kDa to conjugates with molecular weight less than 70 kDa. In contrast, two curves were identical in the zone of 170–70 kDa (Figures 4C and S4B). We suggest that substrates in this area are equilibrated in terms of the amount of Ub moieties per protein. At the same time, Ub is removed by DUBs from the more ubiquitinated substrates and is simultaneously recruited by Ub ligases for ubiquitination of substrates with short polyUb chains. Plotting of intensity of resorufin fluorescence depending on the mass of protein-Ub conjugates after 6 h of incubation with DMSO in comparison with the superposition of curves corresponded to the starting point and 6 h under PS-341 exposure suggests that proteasome hydrolyzes protein-Ub conjugates with a molecular weight of 200–70 kDa. In contrast, a large portion of “super-ubiquitinated” proteins was untouched by the proteasome (Figure 4C). Taking into the consideration that the average molecular weight of protein in the human proteome is 30 kDa (Tran et al., 2011Tran J.C. Zamdborg L. Ahlf D.R. Lee J.E. Catherman A.D. Durbin K.R. Tipton J.D. Vellaichamy A. Kellie J.F. Li M. et al.Mapping intact protein isoforms in discovery mode using top-down proteomics.Nature. 2011; 480: 254-258Crossref PubMed Scopus (503) Google Scholar) (with correction according to their abundance), plotting of differential changes in resorufin fluorescence depending on the mass of the protein-Ub conjugates (Figures 4D and S4C) suggests that dynamic equilibrium between ubiquitination and deubiquitination corresponds to the nearly 6–7 Ub monomers per protein molecule. Overlapping of distribution of human proteome relative to the mass of polypeptides (Tran et al., 2011Tran J.C. Zamdborg L. Ahlf D.R. Lee J.E. Catherman A.D. Durbin K.R. Tipton J.D. Vellaichamy A. Kellie J.F. Li M. et al.Mapping intact protein isoforms in discovery mode using top-down proteomics.Nature. 2011; 480: 254-258Crossref PubMed Scopus (503) Google Scholar) virtually carrying six Ub also nicely fits the same zone of the ubiquitination-deubiquitination equilibrium (Figure S4C). The 12 Ub molecules per protein, representing one more detected local equilibrium point, more likely represent branched or multisite polyubiquitination with two six-length or three four-length Ub chains per one protein rather than a long single one (Lu et al., 2015Lu Y. Lee B.H. King R.W. Finley D. Kirschner M.W. Substrate degradation by the proteasome: a single-molecule kinetic analysis.Science. 2015; 348: 1250834Crossref PubMed Scopus (141) Google Scholar). Our data suggest that polyubiquitinated proteins exceeding 20–25 Ub monomers per protein seem to be penalized by the proteasome, herewith deubiquitination activity toward these conjugates increases logarithmically in accordance with their length (Figure 4D). Each Ub molecule may be linked with the next Ub in a chain through seven different lysine residues or N-terminal methionine. Next, we aimed to analyze the stability of Ub, forming polyUb chains linked preferentially through distinct lysine residues. To elucidate this, we utiliz" @default.
• W3134459642 created "2021-03-15" @default.
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• W3134459642 title "In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking" @default.
• W3134459642 cites W1491060698 @default.
• W3134459642 cites W1496897161 @default.
• W3134459642 cites W1540566915 @default.
• W3134459642 cites W1541511139 @default.
• W3134459642 cites W1553797204 @default.
• W3134459642 cites W1617606969 @default.
• W3134459642 cites W1862346079 @default.
• W3134459642 cites W1960421135 @default.
• W3134459642 cites W1968948718 @default.
• W3134459642 cites W1972434272 @default.
• W3134459642 cites W1977891703 @default.
• W3134459642 cites W1979784352 @default.
• W3134459642 cites W1981426415 @default.
• W3134459642 cites W1984981795 @default.
• W3134459642 cites W1985829088 @default.
• W3134459642 cites W1986429623 @default.
• W3134459642 cites W1992174702 @default.
• W3134459642 cites W1996015633 @default.
• W3134459642 cites W2004552422 @default.
• W3134459642 cites W2006061029 @default.
• W3134459642 cites W2007003700 @default.
• W3134459642 cites W2007452808 @default.
• W3134459642 cites W2008744347 @default.
• W3134459642 cites W2010250927 @default.
• W3134459642 cites W2010643291 @default.
• W3134459642 cites W2012057388 @default.
• W3134459642 cites W2012671659 @default.
• W3134459642 cites W2013339424 @default.
• W3134459642 cites W2016036293 @default.
• W3134459642 cites W2017033636 @default.
• W3134459642 cites W2020392060 @default.
• W3134459642 cites W2023687151 @default.
• W3134459642 cites W2024543003 @default.
• W3134459642 cites W2033149683 @default.
• W3134459642 cites W2035094622 @default.
• W3134459642 cites W2039778506 @default.
• W3134459642 cites W2044843127 @default.
• W3134459642 cites W2052302154 @default.
• W3134459642 cites W2054601514 @default.
• W3134459642 cites W2061050157 @default.
• W3134459642 cites W2063695715 @default.
• W3134459642 cites W2064715535 @default.
• W3134459642 cites W2067488012 @default.
• W3134459642 cites W2069494008 @default.
• W3134459642 cites W2072422417 @default.
• W3134459642 cites W2079228567 @default.
• W3134459642 cites W2080625914 @default.
• W3134459642 cites W2088833470 @default.
• W3134459642 cites W2090682502 @default.
• W3134459642 cites W2091637552 @default.
• W3134459642 cites W2092421259 @default.
• W3134459642 cites W2104192693 @default.
• W3134459642 cites W2108031465 @default.
• W3134459642 cites W2126258150 @default.
• W3134459642 cites W2128296472 @default.
• W3134459642 cites W2128551987 @default.
• W3134459642 cites W2134178604 @default.
• W3134459642 cites W2140946052 @default.
• W3134459642 cites W2143538182 @default.
• W3134459642 cites W2144830324 @default.
• W3134459642 cites W2149954047 @default.
• W3134459642 cites W2154068854 @default.
• W3134459642 cites W2157162286 @default.
• W3134459642 cites W2157193371 @default.
• W3134459642 cites W2162223351 @default.
• W3134459642 cites W2166987747 @default.
• W3134459642 cites W2169284795 @default.
• W3134459642 cites W2170568632 @default.
• W3134459642 cites W2255129447 @default.
• W3134459642 cites W2277539891 @default.
• W3134459642 cites W2321779592 @default.
• W3134459642 cites W2409237344 @default.
• W3134459642 cites W2463195069 @default.
• W3134459642 cites W2469126442 @default.
• W3134459642 cites W2544360569 @default.
• W3134459642 cites W2756668630 @default.
• W3134459642 cites W2762198049 @default.
• W3134459642 cites W2788812105 @default.
• W3134459642 cites W2797999832 @default.
• W3134459642 cites W2887405675 @default.
• W3134459642 cites W2897820323 @default.
• W3134459642 cites W2953285121 @default.
• W3134459642 cites W2958599763 @default.
• W3134459642 doi "https://doi.org/10.1016/j.chembiol.2021.02.009" @default.

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