Assembly Mechanisms of Specialized Core Particles of the Proteasome

The 26S proteasome has a highly complicated structure comprising the 20S core particle (CP) and the 19S regulatory particle (RP). Along with the standard CP in all eukaryotes, vertebrates have two more subtypes of CP called the immunoproteasome and the thymoproteasome. The immunoproteasome has catalytic subunits β1i, β2i, and β5i replacing β1, β2, and β5 and enhances production of major histocompatibility complex I ligands. The thymoproteasome contains thymus-specific subunit β5t in place of β5 or β5i and plays a pivotal role in positive selection of CD8+ T cells. Here we investigate the assembly pathways of the specialized CPs and show that β1i and β2i are incorporated ahead of all the other β-subunits and that both β5i and β5t can be incorporated immediately after the assembly of β3 in the absence of β4, distinct from the assembly of the standard CP in which β-subunits are incorporated in the order of β2, β3, β4, β5, β6, β1, and β7. The propeptide of β5t is a key factor for this earlier incorporation, whereas the body sequence seems to be important for the earlier incorporation of β5i. This unique feature of β5t and β5i may account for preferential assembly of the immunoproteasome and the thymoproteasome over the standard type even when both the standard and specialized subunits are co-expressed.


Introduction
Protein degradation exerted by the ubiquitin-proteasome system (UPS) starts from conjugation of ubiquitin chains to target proteins. Polyubiquitinated proteins are recognized and captured by a huge enzyme complex called the 26S proteasome and are then digested to short peptide fragments [1]. Regulated protein degradation by the UPS is critically involved in various cellular processes such as cell cycle regulation, transcription regulation, and intracellular signaling [2].
The 26S proteasome contains a catalytic core particle (CP; also called the 20S proteasome) and 19S regulatory particles (RP) bound at one or both ends of the CP. The RP contains subunits for capturing ubiquitinated proteins and subunits with ATPase domains for unfolding substrate proteins, thus enabling the CP to degrade proteins [3]. The CP is a cylindrical complex and provides an enclosed cavity in which proteins are degraded [1]. It consists of stacks of four seven-membered rings; two outer Į-rings comprised of Į1-Į7 and two inner ȕ-rings comprised of ȕ1-ȕ7 [4]. The Į-ring serves as docking sites for the RP, and the N-termini of Į-subunits form a gate that regulates access of substrates to the catalytic sites that reside at the inner surface of the ȕ-ring [5]. Of the ȕ-subunits, ȕ1, ȕ2, and ȕ5 exhibit proteolytic activities known as caspase-like, trypsin-like, and chymotrypsin-like activities, respectively [6].
The assembly pathway of the proteasome, which is well conserved in budding yeast and human, is highly complicated, probably due to the large number of its subunits [7,8]. To date, the assembly of the CP has been extensively studied. It has been shown that the assembly of the CP is assisted by dedicated chaperones PAC1-PAC2/Pba1-Pba2 complex, PAC3-PAC4/Pba3-Pba4 complex, and UMP1 (or POMP)/Ump1 in mammals/budding yeast. The N-terminal propeptides and C-terminal tails of some ȕ-subunits also play pivotal roles during the assembly [9][10][11][12][13][14][15][16][17][18]. A complex comprising an Į-ring, PAC1-PAC2, and PAC3-PAC4 is known as the earliest intermediate found in mammalian cells [13,15]. This complex provides a platform for the subsequent assembly of ȕ-subunits. Among the seven ȕ-subunits, ȕ2 assembles on the Į-ring first of all, followed by sequential incorporation of the remaining ȕ-subunits in the order ȕ3, ȕ4, ȕ5, ȕ6, and ȕ1 [19]. The resulting intermediate without ȕ7 is detected as a half-proteasome precursor or half-mer, whose dimerization is driven by the propeptide of ȕ5 and the C-terminal tail of ȕ7 [14,20]. During the ȕ-ring assembly process, PAC3-PAC4 complex dissociates upon ȕ3 incorporation, whereas the PAC1-PAC2 complex stays on the Į-ring until completion of CP assembly. UMP1 serves as an essential chaperone in recruiting ȕ2 and in maintaining the intermediates until a full set of ȕ-subunits are incorporated on the Į-ring [20]. Maturation of CP is accomplished through the processing of the ȕ-subunit propeptides and degradation of UMP1 and PAC1-PAC2 [11,21].
Besides the standard CP, which have ȕ1, ȕ2, and ȕ5 as catalytic subunits, two other types of CP that mainly work in the immune system are found in vertebrates. One is the immunoproteasome, which contains the immune-subunits ȕ1i, ȕ2i, and ȕ5i as catalytic subunits. Its expression is induced by interferon-Ȗ (IFN-Ȗ) or occurs constitutively in immune organs such as the thymus and the spleen [22]. ȕ1i, ȕ2i, and ȕ5i are preferentially incorporated into the CP in place of the corresponding subunits ȕ1, ȕ2, and ȕ5 during the biogenesis of the CP. Another is the thymoproteasome, which contains ȕ1i, ȕ2i, and ȕ5t as catalytic subunits, where ȕ5t is expressed exclusively in cortical thymic epithelial cells (cTECs) [23]. The proteasome plays a central role in the adaptive immune system by producing peptides bound to the major histocompatibility complex (MHC) class I in vertebrates [2]. The immunoproteasome generates more peptides suitable for binding to MHC class I than the standard CP, thus facilitating presentation of foreign antigens to CD8 + cytotoxic T cells. Recently it was reported that the immunoproteasome also works in degrading oxidized proteins [24]. The thymoproteasome carries out a key role in efficient positive selection of the developing CD8 + T cell in the thymus, probably presenting a unique peptide repertoire on the MHC class I molecules of cTECs [23,25].
While the assembly pathway of the standard CP has been studied in detail, those of the specialized CPs are not fully examined. Previous reports have shown that the propeptides of the immune-subunits and UMP1 play key roles in the immunoproteasome assembly [4,20,26]. They also showed mutually dependent incorporation of ȕ1i and ȕ2i. However, the exact order of subunit incorporation is not understood.
In this paper, we dissected ȕ-ring assembly pathway of the immunoproteasome and the thymoproteasome using small interfering RNA (siRNA)-mediated knockdown of ȕ-subunits, which caused accumulation of a specific intermediate before the incorporation of a targeted subunit. By analyzing these intermediates, we clarified the order of ȕ-subunit incorporation on the Į-ring in these specialized CPs. In addition, we investigated the role of the ȕ5t propeptide in the earlier incorporation into the premature CP, which revealed that the propeptide of ȕ5t is a key factor for its earlier incorporation than ȕ4.

ȕ4-Independent Incorporation of ȕ5i on the Į-Ring during Immunoproteasome Assembly
To clarify the assembly pathway of the ȕ-ring of the immunoproteasome, we utilized siRNA-mediated knockdown of each ȕ-subunit of the immunoproteasome. This method worked well for elucidating the assembly mechanism of the standard CP [19]. It is expected that intermediates would accumulate due to the absence of the targeted subunit. We used HeLa cells treated with IFN-Ȗ to induce the immuno-subunits ȕ1i, ȕ2i, and ȕ5i. To observe bona fide assembly pathway of the immunoproteasome, the expression of their homologous counterparts ȕ1, ȕ2, and ȕ5 was repressed by siRNAs 24-h before each knockdown of subunits constituting the immunoproteasome. Accumulated intermediates were characterized by native-PAGE followed by immunoblot analysis for Į6, ȕ1i, ȕ2i, ȕ3, ȕ4, ȕ5i, ȕ6, and ȕ7 ( Figure 1).
Immunoblotting for Į6 revealed a decrease in assembled CP and accumulation of intermediates with various molecular masses in each of the knockdown cells ( Figure 1A). These results indicated that each knockdown caused arrest of the assembly pathway at specific stages and suggested that ȕ-subunits of the immunoproteasome were incorporated on the Į-ring in a sequential manner, just as the assembly of the standard CP [19]. In ȕ4-, ȕ5i-, ȕ6-, and ȕ7-knockdown cells, at least two intermediates with different masses were observed. The faster migrating bands were PAC1-PAC2 associated forms, which appeared as doublets in lanes of ȕ5i and ȕ7 RNAi for unknown reason, and the slower migrating bands were PA28 associated forms ( Figure 2E,G,H).
In the standard CP assembly, ȕ2 is the first subunit assembled on the Į-ring. However, ȕ1i and ȕ2i are likely to be incorporated on the Į-ring ahead of the other ȕ-subunits in the immunoproteasome assembly, because intermediates accumulated in ȕ1i-and ȕ2i-knockdown cells shared the same molecular mass with the control cells, which only contained the Į-ring [12] ( Figure 1A). This was further supported by the observation that ȕ1i and ȕ2i were detected in all the intermediates except for those in their own knockdown ( Figure 1B,C) and that the intermediates that accumulated in ȕ1i-and ȕ2i-knockdown cells did not contain any other ȕ-subunits ( Figure 1D-H, see lanes of ȕ1i and ȕ2i RNAi). These results indicate that simultaneous incorporation of ȕ1i and ȕ2i is necessary as the first step of ȕ-ring assembly of the immunoproteasome. This view is consistent with the previous finding that ȕ1i and ȕ2i are mutually required for their incorporation during the immunoproteasome assembly [27].  The assembly of ȕ3 followed that of ȕ1i and ȕ2i, given that ȕ3 was identified in the intermediates of cells treated with siRNA targeting ȕ4, ȕ5i, ȕ6, and ȕ7 ( Figure 1D), and therefore the incorporation of ȕ3 should precede these subunits. Consistent with this view, the intermediate of ȕ3-knockdown cells contained ȕ1i and ȕ2i, but not ȕ4, ȕ5i, ȕ6, and ȕ7 ( Figure 1B,C,E,H).
Either ȕ4 or ȕ5i can be incorporated on the Į-ring immediately after the incorporation of ȕ3, because ȕ4 was detected in the ȕ5i-knockdown intermediates ( Figure 1E, lane of ȕ5i RNAi), and ȕ5i was also recognized in the ȕ4-knockdown intermediates ( Figure 1F, lane of ȕ4 RNAi). The ȕ4-independent incorporation of ȕ5i was in marked contrast to the incorporation of ȕ5 during the standard CP assembly, which required the preceding assembly of ȕ4 on the Į-ring [19].
ȕ6 is recruited after both ȕ4 and ȕ5i were assembled on the Į-ring, as evidenced by the presence of ȕ6 only in the intermediates of ȕ7-knockdown cells ( Figure 1G) and the presence of all the ȕ-subunits other than ȕ6 and ȕ7 in the intermediates of ȕ6-knockdown cells ( Figure 1B-H). ȕ7 is the last ȕ-subunit incorporated in the pre-immunoproteasome because ȕ7 was not found in any of the intermediate complexes ( Figure 1H), consistent with the former reports on the assembly pathway of the standard CP [19].
To sum up, the order of ȕ-subunit assembly of the immunoproteasome is different from that of the standard CP in two points; one is the simultaneous incorporation of ȕ1i and ȕ2i as the first step, and the other is ȕ4-independent incorporation of ȕ5i.

Conserved Roles of Assembly Chaperones during Immunoproteasome Biogenesis
The assembly of the standard CP is assisted by proteasome-dedicated chaperones UMP1, PAC1-PAC2 complex, and PAC3-PAC4 complex, each of which plays a specific role [8]. To know whether their roles and molecular behavior in immunoproteasome assembly are the same as those in the standard CP assembly, we examined in which intermediates these chaperones were included. UMP1-knockdown cells accumulated intermediates with the same mass as the intermediates of ȕ2i-knockdown cells (Figure 2A) and failed to incorporate ȕ1i and ȕ2i ( Figure 2B,C). Furthermore, without ȕ2i, UMP1 was not present on the intermediates ( Figure 2D). These results suggest that the initial incorporation of ȕ1i and ȕ2i depends on UMP1 and vice versa, similar to interdependent incorporation of ȕ2 and UMP1 into the standard CP [19].
PAC1 was detected in the intermediates of ȕ1i-and ȕ2i-knockdown cells and the faster migrating intermediates of other ȕ-subunit knockdown cells ( Figure 2E). This is the same as its role in the standard CP assembly; PAC1 not only helps efficient Į-ring assembly and prevents its dimerization, but also continues to associate with the Į-ring until all the ȕ-subunits incorporated into the CP [19].
PAC3 associated with intermediates of ȕ1i-, ȕ2i-, and ȕ3-knockdown cells and was absent from intermediates in cells where the ȕ-subunits incorporated after ȕ3, i.e., ȕ4, ȕ5i, ȕ6, and ȕ7, were knocked down ( Figure 2F). Therefore, the release of PAC3 is coupled with the incorporation of ȕ3 in the immunoproteasome assembly, which is the same timing as in the standard CP [19].

Earlier Incorporation of ȕ5i Is Independent of ȕ1i and ȕ2i
As shown in Figure 1, ȕ5i can be incorporated on the Į-ring ahead of ȕ4 in precursor immunoproteasomes. This is in marked contrast to ȕ5 incorporation into precursors of standard CPs, which requires preceding incorporation of ȕ4 on the Į-ring [19]. In order to clarify whether this earlier incorporation of ȕ5i than ȕ4 depends on ȕ1i and ȕ2i and whether the standard subunit ȕ5 can also be incorporated before ȕ4 in the presence of ȕ1i and ȕ2i, IFNȖ-treated HeLa cells were knocked down in the combinations shown in Figure 3A. The cell lysates were separated by native-PAGE, followed by immunoblot analysis using antibodies to ȕ5i and ȕ5. Consistent with the results shown in Figure 1, ȕ5i was incorporated into the intermediates comprised of Į-ring, ȕ1i, ȕ2i, and ȕ3 in the absence of ȕ4 ( Figure 3B, lane 2). Also, consistent with the previous report [19], ȕ5 was not incorporated in the intermediates comprised of Į-ring, ȕ2, and ȕ3 in the absence of ȕ4 ( Figure 3C, lane 5). Even in the presence of ȕ1i and ȕ2i, ȕ5 failed to be incorporated in the intermediate without ȕ4 (Figure 3C, lane 3), suggesting that preceding incorporation of ȕ1i and ȕ2i was not a determinant of earlier incorporation of ȕ5-type subunits. Rather, ȕ5i can be incorporated in the intermediate without ȕ4 (Figure 3B, lane 4).  Thus, the ability of ȕ5i to assembly on the Į-ring without preceding presence of ȕ4 is intrinsic to ȕ5i itself and does not depend on ȕ1i and ȕ2i.

ȕ5t Can Also Be Incorporated before ȕ4 during CP Assembly
ȕ5t is specifically expressed in cTECs of the thymus, where it occupies the majority of the ȕ5 positions in the CP, despite co-expression of ȕ5i at the mRNA level [28]. At present, there is no available cell line that expresses endogenous ȕ5t. Therefore, we established a HEK293T-derived cell line stably expressing human ȕ5t with C-terminal Flag-tag (hereafter referred to as ȕ5t-Flag cell) to ask if ȕ5t employs a unique assembly strategy. HEK293T cells do not express immuno-subunits at all, and the assembly pathway of the standard CP in this cell line is well-studied, as described previously [19].
To examine how ȕ5t-containing CP is assembled, we performed knockdown of ȕ1, ȕ2, ȕ3, ȕ4, ȕ5t, ȕ6, and ȕ7, each along with ȕ5. Immunoblot analysis following native-PAGE of the cell lysates showed accumulation of different intermediate complexes in each knockdown ( Figure 4A), similar to the analysis of the immunoproteasome assembly and the standard CP assembly [19]. Immunoblot for ȕ4 and Flag (ȕ5t) revealed that either ȕ4 or ȕ5t can be incorporated on the Į-ring immediately after the incorporation of ȕ3, as evidenced by the observation that ȕ4 and ȕ5t was detected in the ȕ5t-and ȕ4-knockdown intermediates, respectively ( Figure 4B, lane of ȕ5t RNAi, and Figure 4C, lane of ȕ4 RNAi). Therefore, ȕ5t can be also incorporated in the intermediate comprised of Į-ring, ȕ2, and ȕ3 that does not include ȕ4. This ȕ4-independent incorporation of ȕ5t is quite similar to the incorporation of ȕ5i and in marked contrast to the incorporation of ȕ5.

Role of the Propeptides of ȕ5i and ȕ5t in the Earlier Incorporation
As shown in Figure 3 and Figure 4, both ȕ5i and ȕ5t can be incorporated on top of the Į-ring during the CP assembly at an earlier stage than ȕ4 incorporation, and these abilities were not dependent on ȕ1i and ȕ2i and appeared to be intrinsic to ȕ5i and ȕ5t. Both ȕ5i and ȕ5t are synthesized as precursor proteins comprised of a propeptide portion and a mature portion. The propeptide portion is processed upon completion of the CP assembly. Since the propeptide of ȕ5 is known to play an important role in the incorporation of ȕ5 during the CP assembly [26], we next examined whether the unique feature of ȕ5i and ȕ5t is dependent on their propeptides.
Mutant ȕ5 subunits with C-terminal Flag-tag, in which the propeptide portions were replaced by the propeptide of ȕ5i or ȕ5t (referred to as ȕ5i (p) + ȕ5 (m) and ȕ5t (p) + ȕ5 (m), respectively), were expressed in HEK293T cells. To see whether the mutant ȕ5 subunits can be incorporated without ȕ4, the presence of the mutant ȕ5 subunits was examined in the intermediates that were accumulated by knockdown of endogenous ȕ4 by native-PAGE followed by immunoblot analysis for Flag ( Figure 5). These mutant ȕ5 subunits were readily incorporated into the complete CPs ( Figure 5; lane 1, 4, and 7). When ȕ6 and endogenous ȕ5 were knocked down, intermediates during CP assembly were accumulated, where the mutant ȕ5 subunits were incorporated instead of endogenous ȕ5 ( Figure 5; lane 2, 5, and 8). When ȕ4 was knocked down, the wild-type ȕ5 was not detected in the assembly intermediates ( Figure 5; lane 3). ȕ5i (p) + ȕ5 (m) also failed to be incorporated in the absence of ȕ4 ( Figure 5; lane 6), suggesting that the propeptide of ȕ5i is not responsible for ȕ4-independent ȕ5i incorporation, rather suggesting that the mature portion of ȕ5i enables it. In contrast, ȕ5t (p) + ȕ5 (m) was readily incorporated in the assembly intermediates without ȕ4 ( Figure 5; lane 9), suggesting that the propeptide of ȕ5t is sufficient for ȕ4-independent ȕ5t incorporation.

Maturation of ȕ5t Is Largely Dependent on IFN-Ȗ
As shown in Figure 3, ȕ5i can be incorporated immediately after ȕ3, and this ability did not depend on ȕ1i and ȕ2i. Since ȕ1i and ȕ2i are the common catalytic subunits of the immunoproteasome and the thymoproteasome, we then examined whether there was any difference in the dependence of incorporation of ȕ5i and ȕ5t on the presence of ȕ1i and ȕ2i. We expressed ȕ5t or ȕ5i in ȕ5i-deficient MEF cells. These cells express ȕ1i and ȕ2i only when treated with IFN-Ȗ. Nearly half of the expressed ȕ5t were in premature forms without IFN-Ȗ, but the mature ȕ5t was remarkably increased upon IFN-Ȗ treatment ( Figure 6, IB of ȕ5t). In contrast, the majority of ȕ5i were already matured in the absence of IFN-Ȗ, and the induction of ȕ5i maturation by IFN-Ȗ was modest ( Figure 6, IB of ȕ5i). These results suggest that the presence of ȕ1i and ȕ2i facilitated incorporation of ȕ5t, whereas ȕ5i was incorporated efficiently in combination with the standard subunits ȕ1 and ȕ2. Alternatively, it may also be possible that the propeptide of ȕ5i is processed more efficiently by ȕ1i and ȕ2i. Maturation of ȕ1i and ȕ2i was facilitated not only by the presence of ȕ5i, which was already known [27], but also by the presence of ȕ5t ( Figure 6, IB of ȕ1i and ȕ2i), suggesting the interdependent maturation of ȕ1i, ȕ2i, and ȕ5t.

Discussion
Making use of HeLa cells treated with IFN-Ȗ, we clarified the assembly pathways of ȕ-subunits of the immunoproteasome ( Figure 7A). Beginning with the simultaneous incorporation of ȕ1i, ȕ2i, and UMP1 on the Į-ring, the adjacent ȕ-subunits assembled sequentially in a defined order. A similar assembly pathway was observed during the formation of the thymoproteasome. This is in contrast to the standard CP assembly, where ȕ1 is the last but two ȕ-subunit incorporated ( Figure 7B). An intermediate containing ȕ1i, ȕ2i, ȕ3, and ȕ4 has been reported previously [10], where ȕ1i plays an important role in the assembly of the immunoproteasome [27]. Our results support the view that the early incorporation of ȕ1i is required for the initiation of the immunoproteasome biogenesis. Figure 7. Assembly pathways of the immunoproteasome, the thymoproteasome, and the standard CP. (A) Assembly pathway of the immunoproteasome and the thymoproteasome started with incorporation of ȕ1i and ȕ2i, followed by ȕ3 and ȕ4. Both ȕ5i and ȕ5t can also be incorporated immediately after ȕ3. ȕ6 and ȕ7 were the last two subunits to be incorporated; (B) Assembly pathway of the standard CP for reference.
We also observed that ȕ5i and ȕ5t can be incorporated immediately after ȕ3 incorporation and in a ȕ4-independent manner. This is in marked contrast to incorporation of ȕ5 into the standard CP, which is dependent on ȕ4. In the standard CP assembly, ȕ5 is incorporated after the formation of a "13S complex" composed of Į-ring, ȕ2, ȕ3, and ȕ4. Previous reports have shown that overexpression of ȕ5 increases the amount of mature CP [18,29]. This suggests that incorporation of ȕ5 is a rate-limiting step during the CP assembly. The earlier incorporation of ȕ5i and ȕ5t might play some role in preferential formation of the immunoproteasome and the thymoprotesome over the standard proteasome.
It is also intriguing that more than 90% of the CP is the thymoproteasome in cTECs, although ȕ5t and ȕ5i are transcriptionally co-expressed. We showed that the propeptide of ȕ5t but not that of ȕ5i is sufficient for the ȕ4-independent incorporation. Furthermore, incorporation of ȕ5t seems to be more dependent on ȕ1i and ȕ2i than that of ȕ5i, because maturation of ȕ5t was greatly enhanced by IFN-Ȗ, compared to that of ȕ5i. These features of ȕ5t may explain the predominant expression of the thymoproteasome over the immunoproteasome in cTECs.

Cell Culture
Cells were cultured as described previously [19]. For induction of immuno-subunits, cells were cultured in the presence of 50 U/mL IFN-Ȗ (Peprotec, Rocky Hill, NJ, USA) and incubated for 48 h. Plasmid transfection was performed using Lipofectamine2000 (Thermo Fisher Scientific Inc. Waltham, MA, USA), and cells were selected with 4 ȝg/mL puromycin (Sigma Aldrich, St. Louis, MO, USA) to obtain stable transfectants.

DNA Constructs
Plasmids encoding ȕ5i (p) + ȕ5 (m) and ȕ5t (p) + ȕ5 (m) were constructed by fusing cDNAs encoding the propeptides of ȕ5i and ȕ5t to the 5' end of the cDNA encoding mature form of ȕ5, respectively. PCR was performed using PrimeSTAR Max DNA Polymerase (TaKaRa Bio Inc. Shiga, Japan). The cDNAs were subcloned into pIRESpuro3 vector. Synonymous mutations were introduced to confer resistance to siRNAs. All constructs were confirmed by sequencing.

RNA Interference
The siRNAs targeting human ȕ-subunits and UMP1 (Table 1) were transfected into HeLa cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc.) at a final concentration of 50 nM. For each sample, 9 × 10 5 cells were plated in a 100-mm dish six hours before transfection. Transfected cells were incubated for 36 h before the analysis.