Figure 1.
A–C. Cos7 cells were treated with DMSO (A) or treated with 5 µM MG132 for 16 hours (B–C) and cells were stained with a monoclonal mix of anti-ubiquitin (A and B), the anti-p300 polyclonal C20 antibody (A–C), or anti-vimentin monoclonal (C) antibodies (see materials and methods for details on the antibodies). The arrow in panel A, indicates the presence of p300 in diffused cytoplasmic aggregates. The arrows in B and C indicate the position of representative aggresomes, enclosed by vimentin and containing ubiquitin. Cells were subjected to fluorescence deconvolution and image reconstruction by using a Zeiss microscope (Axiovert 200M) with deconvolution capabilities (Axiovision 4.1). The merge panels represent the deconvoluted images from the Alexa-Fluor 488 (green), Alexa-Fluor 568 (red), and DAPI signals. D. Quantification of the immuno-fluorescence experiments. The percentage of cells containing aggresomes (black bars) and of aggresomes containing p300 (gray bars) was calculated in mock treated (−) or MG132 treated (+) cells. E. Cos7 cells were mock treated (−; lanes 1 and 4) or treated with MG132 (+; lanes 2, 3 and 5) and cell extracts were subjected to immuno-precipitation with a control antibody (lane 3) or with the anti-p300 antibody (lanes 4 and 5). Immuno-precipitation reactions were divided into different aliquots and subjected to immuno-blot with anti p300 (top panel), anti-HDAC6 (mid panel) or anti dynein antibodies (bottom panel), as indicated. Lanes 1 and 2 contains inputs levels of the indicated proteins.
Figure 2.
p300 co-localizes with a-synuclein, ubiquitin and HDAC6 in brain of patients affected by Parkinson Disease.
Sections of midbrains or cortex from normal control patients (A), or from patients by Parkinson Disease (B–E) were immuno-stained with the anti-p300 (red) and α-synuclein (green, panels B,C), or with anti-ubiquitin (green, panel D), or with anti-HDAC6 (green, panel E) antibodies as indicated at the top of each panel. Different sections from the same patients or from different patients were subjected to analysis.
Figure 3.
Identification of the structural domain of p300 responsible for localization into aggresomes.
A. Schematic representation of the various p300 domains (see also text for further description). The three Cystein-Histidine-Rich domains, (CH1, CH2 and CH3), are delineated by colored boxes. The bromodomain (Br) and histone acetylatransferase domains (HAT) are labeled by colored horizontal lines. Under the diagram, a scheme of the p300 expression vectors and relative amino acid residues affected by deletions/mutations used for detection of p300 in aggresomes, is shown. B. Quantification of the immuno-fluorescence experiments, showing the percent of cells containing p300 in aggresomes relatively to the total number of cells expressing each of these deletion products. Cos7 cells were transfected with the vectors expressing p300 full-length, p300Δ30 or p300-N, p300-Δ242–1737 or p300-CH3-CTD and the percentage of cells containing p300 in aggresomes was calculated from two separate experiments. Representative immuno-fluorescence data from these experiments are shown in panel C. C. Cells were treated with MG132, and subsequently stained for vimentin (red), or for p300 and then counterstained with DAPI (only shown in the merge panels). Arrows indicate the position of aggresomes.
Figure 4.
In silico analysis of the region of p300 responsible for the localization into aggresomes. A.
Blast results of the region of p300 encompassing amino acid residues 1514 to 2414 showing representative similarities with the prion-like protein pqn-85 of C.Elegans. The results of this search are displayed in more detail in table S1. Amino acid residues labeled in red designate identity, while the blue color outlines the Q and N residues in the p300 or pqn-85 proteins. p300 has a 22% total content of Q/N in this region B. Compositional analysis of full length p300 (red) or of the p300-CTD (green) in comparison with composition of typical ordered proteins. Compositional profile of typical intrinsically disordered proteins from the DisProt database is shown for comparison (black bars). Positive bars correspond to residues found more abundantly in p300, while negative bars show residues, in which p300 is depleted. This analysis shows that compared to ordered proteins, p300 is enriched in the disorder-promoting residue C, H, M, Q, S, N, and P and is depleted in major order-promoting residues W, F, I, Y, V, L, A, R, D, E and K. Furthermore there is a low content of charged residues, with enrichment in polar but non-charged residues. C–D. Evaluation of intrinsic disorder in the full-length p300 (C) and its C-terminal domain (CTD), residues 1514–2414 (D). Four disorder prediction tools of the PONDR family were used (28–30): PONDR® VSL2B, which is statistically better for proteins containing both structure and disorder; PONDR® VL3 which is better for proteins that are experimentally known to be 100% disordered or possess long disordered regions; PONDR® VLXT which is useful for predicting MoRFs, short disordered regions that become structured when they interact with their binding partners; and PONDR-FIT a meta-predictor which is statistically not different from PONDR® VL3 for fully disordered and fully structured proteins, and slightly better (1 std) than PONDR® VSL2 when both structured and disordered regions are present. The dark gray lines are disorder predictions by PONDR®VLXT; the red lines represent results of disorder prediction by PONDR®VL3; the blue lines show disorder predictions by PONDR®VSL2; whereas green lines correspond to the results produced by PONDR-FIT; light green shadows represent standard errors of disorder prediction by PONDR-FIT. Locations of structured domains (CH1 domain, residues 323–423 (PDB IDs: 1L3E and 1P4Q); bromo-domain, residues 1040–1161 (PDB ID: 3I3J); acetyltransferase domain (HAT), residues 1284–1669 (PDB ID: 3BIY); and TAZ2 domain, residues 1723–1836 (PDB ID: 3IO2) are shown by dark blue and cyan bars. Figure 4C and 4D show that these proteins are highly disordered, since their curves are mostly located above 0.5, albeit they also contain ordered regions. Figure 4C also shows that p300 contains a large number of potential disorder-based binding sites, α-MoRFs and ANCHOR-indicated binding sites (AiBSs) (29–30). Locations of α-MoRFs and AiBSs are shown as pink and dark cyan bars at the bottom of plots.
Figure 5.
An alternatively spliced product of p300 encompasses the PSPD region and is enriched in MG132 treated cells.
A. Schematic representation of the alternative exons and spliced variants of p300. These data were extracted from the Aceview database (31,32). B. Structure of the pre-messenger mRNA (variant b, http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&term=EP300&submit=Go). This complete CDS mRNA is 3297 base pair long. It is predicted to encode a protein of 85 kDa which is identical in sequence to the full length p300, encompassing amino acid residues 1624 to 2414. To gain evidence for the presence of this mRNA, we employed a combination of primers localized in the alternative exon (depicted in panel B). As shown in panel C (left panel), an product of expected size is detected in Hek293 cells after reverse transcription using oligo-dT primer 5′. This PCR product was sequenced and matched the sequences of the p300 mRNA. The right panel in C, shows a control amplification reaction with GADPH primers. The position of the amplified products relatively to the 100 bp ladder is indicated. D–E. Detection of a p300 polypeptide lacking the N-terminal region and encompassing the p300 C-terminus. Panel D illustrates the strategy employed for these experiments. Cellular extracts derived from Hek293 untreated or MG132 treated were equalized for protein concentration, and first immuno-precipitated with the anti-p300 antibody directed against the C-terminal region (C20, SC). The product of these immuno-precipitation reactions were then probed in immuno-blot with an antibody directed against either amino acid residues 1572–2371 (RW109; indicated as IB-1), or with antibody raised against the N-terminal region (N15; indicated as IB-2). As shown in panel E, a p300 product of compatible size is detected and enriched in MG132 treated cells in the IB-1 immuno-blot (left panel), but not in the IB-2 IB (mid panel). The right panel shows the inputs levels of cell extracts employed for the immuno-precipitations with an equal actin signal. Arrows indicate the presence of p300 full length or of the PSPD.
Figure 6.
The p300 PSPD is sufficient for aggresome localization.
A–D. Cell transfected with the vector expressing RFP alone and treated with MG132 (panel A), or transfected with the RFP-PSPD (panels B–D) were mock treated (B), or treated with MG132 (C–D) and subsequently stained for vimentin (green). Panels C and D contain two representative fields of cells transfected with RFP-PSPD. Arrows indicate the position of aggregates formed in the absence (panel B) or in the presence of MG132 (C and D). E. Interaction of p300 with ubiquitinated protein species. The epitope-flagged vector expressing the PSPD (lanes 1–5), or the TAZ2 (lanes 6,7 and 10,11) or the ZZ (lanes 8,9 and 12,13) were transfected in Cos7 cells. Cells were left untreated (−) or treated with MG132 (+), cell extracts were immuno-precipitated with the anti-Flag antibody (lanes 1,2; and 6-to-9), or with a control isotype matched antibody (lane 3) and the products of these immuno-precipitation reactions were probed with ubiquitin antibodies as indicated at the bottom of each panel. Alternatively, the anti-Flag immuno-blot on total cell extracts shows the total amount of p300 proteins present in these reactions, which were derived from approximately 1/50 of total extracts.
Figure 7.
A-B. The p300-PSPD brings p53 in cytoplasmic aggregates.
Hek293 cells were co-transfected with a vector expressing epitope tagged GAL4-PSPD and p53. In panel A cells were stained with an antibody recognizing p53 (polyclonal goat, blue), with anti-GAL4 antibody (monoclonal mouse, green), and with the anti-20S proteasome polyclonal antibody (red). The white or red arrows indicate cell expressing or not p300-PSDP. Note that p53 forms inclusions only when p300-PSDP is expressed. In panel B, a vector expressing GFP-p53 was employed to demonstrate co-localization with the 20S subunit of the proteasome (red). Merged images are shown in the last right panel. p53 did not form inclusions with control vector alone (not shown). C. A549 cells e were transfected with scramble, control siRNA (lanes 1 and 3), or with the p300 specific siRNA (lanes 2 and 4). Seventytwo hours after transfection cells were treated with vheicle (lanes 1, 2) or with 20 µM MG132 (lanes 3,4) for 16 hours, and cell extracts were prepared. Total levels of p53 and Hsp70 are shown. D. TOV cells expressing the tumor-derived p53 mutant, p53R175H, were transfected with scramle or p300 shRNA as described in C, and cell extracts derived from these transfection were probed for p53, p300 and actin. E–F. The knock-down of p300 impairs aggresome formation. E. A549 cells were transfected with scramble siRNA or with the p300 specific siRNA. Cells were grown on glass cover slips, treated with MG132 for 16 hours, and then probed with the anti-p300 specific polyclonal antibody (red), or with the monoclonal antibody directed against vimentin (green). Representative fields are shown in D. F. Quantification of the experiments shown in E. Aggresomes were counted in cells transfected with scramble- or p300-specific siRNA and percentages are shown at the top of the bars. Black and gray bars indicates the percentage of cells displaying aggresomes in the presence of control or p300 siRNA. The presence of aggresomes was assessed based on the presence of the vimentin ring. Results are representative of two independent experiments, each performed in duplicate. G. Assessment of HDAC6 expression levels in A549 cells transfected with control- and p300-siRNA.
Figure 8.
Effects of p300 on the misfolded protein response. A.
A549 cells were transfected with scramble siRNA or with the p300 specific siRNA as described previously, and stained with the anti-ubiquitin (green) or anti-vimentin (red) antibody. The percentage of cells containing ubiquitin aggregates is quantified in panel B. C. A549 cells were transfected with the scramble- or p300-specific siRNAs were mock treated or treated with MG132 for 16 hours, then harvested for flow cytometry. Panel C shows the Propidium Iodide profiles, and percentages of cells in the G1 or G2 phases of the cell cycle are indicated at the top of each relative peak. D. A549 cells transfected with scramble (black bars) or with the p300 siRNAs (gray bars), were treated with MG132. In one set of samples MG132 was washed out after 12–14 hours of treatment (indicated as MG132 w/o) and cells were allowed to recover for about four days, at which time they were counted. Alternatively, cell growth was monitored for the same period of time in the presence of MG132 (indicated as MG132 on). Error bars represent standard deviations. E–F. Control (wt) or p300 null mouse embryo fibroblasts (MEF) were mock treated or treated with MG132 for 16–24 hours (panel E), or alternatively, subjected to Heat Shock (HS, panel F) by incubating the cells at 40°C for two hours. Cells were allowed to recover from MG132 treatment or HS for 24–48 hours and were counted. Cell viability was assessed with trypan blue exclusion.
Figure 9.
A. Proposed model for aggregation of the TAZ2 domain depending upon the concentration of zinc.
See also text for explanation. The 1H-15N HSQC spectrum of the TAZ2 apoprotein at low concentration of zinc is poorly dispersed and has narrow line-widths. In low concentration of zinc, the cysteines residues in the fingers will be exposed to solvent in order to avoid intramolecular disulfide bridges. These exposed cysteines with histidines from two chains could interact with zinc and form interchain aggregates. Additional interchain stability could be obtained by forming interchain disulfide bridges between unligated cysteines residues as well as by forming two interchain hydrogen bonds among the side chains of glutamine and asparagine residues which are present throughout the sequence. A schematic representation of intermolecular aggregation is shown. B. Based on data presented here, and in keeping with previous studies that implicated p300 as an E4-ubiquitin ligase, as well as with evidence that p53 is activated by p300 when acetylated, we envision that p300 plays versatile and multiple effects on p53 activity. p53 interacts with at least three sites on p300, as indicated in the Figure, and each interaction site leads to different outcomes. While the interaction with the N-terminus serves to promote degradation of natively folded forms of p53 and to maintain low p53 levels in normal cells, the p300 PSPD brings p53 into cellular aggregates.