Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

Abstract

Background: Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form.

Results: To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells.

Conclusions: It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

Figure 1

Effect of proteasome inhibition by PSI on both cell survival and formation of PSI-induced inclusions in PC12 cells, and assessment of a three-process fractionation procedure on isolation of PSI-induced inclusions from the cells. A, trypan blue staining of cells: a, cell survival (open arrows) alone in cells exposed to DMSO vehicle for 48 h; b, cell survival and addition of cell death (closed arrows) in cells exposed to 10 μM PSI for 48 h; c, percentages of the number of living cells. B, H&E histological staining of inclusions: a, cells in the vehicle; b, nucleus-binding (open arrows) and nucleus-free (closed arrows) inclusions formed in cells in 10 μM PSI for 48 h; c, the two morphological types of inclusions in the fraction of initial pellets; d, percentages of nucleus-free to total inclusions in the cells in PSI (Column 1) and in the fraction of initial pellets (Column 2); e-h, the one (or two) morphological type (s) of inclusions in the first four temporary fractions of resulting pellets; i, the one morphological type of inclusions in the final fraction of resulting pellets; j, percentages of nucleus-free to total inclusions in the five fractions of resulting pellets (Column 3-7). C, alpha-SYN immunostaining for and H&E histological staining of inclusions: a and a', cells in PSI; b and b', an isolated fraction of inclusions. D, immunoblotting for histone H1 in inclusions: a, bands of histone H1 in gel image; b, quantitative level of histone H1 in densitometry. Scale bar, 10 μm.

Figure 2

Coomassie blue staining of proteins resolved in 2-D gel and identification of P4HB via PMF. A, representative patterns of protein spots: a, cells in vehicle; b, cells in PSI; c or d, the isolated fraction of PSI-induced inclusions before and after protein gel spot picking from 2-D gel. B, identification of P4HB via PMF: a, an expanded full scan of PMF; b, the number of theoretically matched peptides and their sequences after preliminary identification via PMF against the single NCBInr database; c-e, comparable values of Mowse score after consistent identification via PMF against the multiple NCBInr, SwissProt, MSDB databases. Mowse score is -10×Log (P), where P is the probability that the observed match is a random event. Significance levels in the multiple databases are 61 in NCBInr branch, 51 in SwissProt branch and 56 in MSDB branch, respectively (p < 0.05). Close arrows indicate locations of the protein spots of interest in 2-D gel for identification, and theoretical sequences of the matched peptides for P4HB after identification.

Figure 3

Functional categories of proteins identified in PSI-induced inclusions. This pie chart shows that all the identified proteins are classified into at least nine accepted categories as follows: 3.6% for synaptic function and neurotransmission (I); 8.8% for gene transcription (II); 3.6% for protein trafficking (III); 21.4% for mitochondrial function and metabolism (IV); 1.8% for antioxidant defense mechanisms (V); 5.4% for apoptotic signal transduction (VI); 10.7% for ubiquitin dependent protein degradation (VII); 30.4% for protein folding and transport (VIII); and 14.3% for unknown function (IX). When a protein could be divided into more than one class of functions, it was indicated as the best known class.

Figure 4

P4HB immunostaining for and H&E histological staining of PSI-induced inclusions developed in PC12 cells after proteasome inhibition by PSI. A and A', cells in the vehicle; B-E and B'-E', cells in PSI for 12, 24, 36 and 48 h; F and F', a fraction of inclusions isolated from the cells in PSI for 48 h. Development of inclusions in the cells at the end of indicated points of PSI exposure is indicated as closed arrows, and nuclei and juxta-inclusion nuclei indicated as closed arrow heads. Scale bar, 10 μm.

Figure 5

Potential mechanisms by which constituents of the UPS and chaperone proteins may affect development of PSI-induced inclusions in PC12 cells after proteasome inhibition by PSI. A, relevance of chaperone proteins in cytoplasm, ER and mitochondria resources to cytoplasmic constituents of the UPS establishes multiple routes of the UPS to remove aberrant and potentially cytotoxic proteins [2,87]. The interaction between constituents of the UPS and cytoplasmic chaperon proteins might present one route to protein degradation in cytoplasm, UPP by which its target proteins serving as protein substrates of enzyme in the cytoplasm are directed by chaperone proteins to proteasomes [2]. The coordination between constituents of the UPS and ER chaperone proteins might present another route to protein degradation in ER, ERAD by which its target proteins serving as protein substrates of enzyme in lumen of the ER are bound to chaperone proteins, retro-translocated through a multi-protein translocon complex across ER membrane, get ubiquitinated by ubiquitin ligases, and are subsequently targeted for degradation by proteasomes [87]. New data support the notion that a route of the UPS similar to that observed in ERAD occurs in mitochondria and therefore the name of MAD has been proposed [87]. B, constituents of the UPS in cytoplasm and chaperone proteins in cytoplasm, ER and mitochondria resources, together with the protein substrates of the UPS, could be recruited to PSI-induced inclusions formed in PC12 cells after proteasome inhibition by PSI.