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(Circulation Research. 2006;99:362.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Mapping the Murine Cardiac 26S Proteasome Complexes

Aldrin V. Gomes, Chenggong Zong, Ricky D. Edmondson, Xiaohai Li, Enrico Stefani, Jun Zhang, Richard C. Jones, Sheeno Thyparambil, Guang-Wu Wang, Xin Qiao, Fawzia Bardag-Gorce, Peipei Ping

From the Departments of Physiology and Medicine (A.V.G., C.Z., X.L., J.Z., G.-W.W., X.Q., P.P.), Cardiac Proteomics and Signaling Laboratory at Cardiovascular Research Laboratories, University of California–Los Angeles; the Food and Drug Administration National Center for Toxicological Research (R.D.E., R.C.J., S.T.), Jefferson, Ark; the Department of Anesthesiology (E.S.), Division of Molecular Medicine, University of California–Los Angeles; and the Departments of Pathology and Medicine (F.B.-G.), Harbor–UCLA Medical Center, Torrance, Calif.

Correspondence to Peipei Ping, Department of Physiology, UCLA School of Medicine, MRL Building, Suite 1609 CVRL, 675 CE Young Dr, Los Angeles, CA 90095. E-mail pping{at}mednet.ucla.edu

See related article, pages 372–380

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species- and cell-type–specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key and ß subunits in cardiomyocytes. The expression of 14 constitutive and ß subunits in parallel with their three inducible subunits (ß1i, ß2i, and ß5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (2, 5, 7, ß3, and ß4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, 7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium.

Key Words: organelle proteomics • 19S and 20S proteasomes • protein degradation • heart

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The proteasome is a key proteolytic enzymatic system governing the degradation of majority intracellular proteins.¹ Recent studies implicate proteasomes in cardiac diseases.^2–11 Proteasome inhibitors are widely used in cancer therapy, however, their impact on cardiac function remains unclear; investigations in the heart have reported conflicting results.^2–4,12 A major contributing factor to these controversies is the lack of information pertaining to the structural organization and protein composition of cardiac proteasomes, therefore prohibiting the identification of molecular targets that these inhibitors intervene in the heart.

Information gained with the yeast model system suggests that the 26S proteasome is composed of subunits arranged into a 20S core complex (14 subunits) and 1 or 2 19S regulatory complexes (up to 17 subunits).^13–15 The 20S proteasome consists of 2 copies of 7 and 7 ß subunits with 2 rings sandwiched with the 2 ß rings.^16–18 The ß1, ß2, and ß5 subunits have distinct proteolytic preference of peptides, namely caspase-like, trypsin-like, and chymotrypsin-like, respectively. The subunits initiate the multimerization of the 20S proteasome, control the entry of substrates, and recruit 19S to the 20S to form 26S proteasome.^1,19,20 In contrast, little is known about 19S proteasomes; there appear to be striking variations in their molecular compositions in various species with the exception of the 6 ATPase subunits, which are highly conserved²¹; they coordinate the activity of 19S and translocate ubiquitinated proteins into the core 20S particle.

The highly organized structure of proteasome complexes may offer multiple sites for functional regulation.²² One key mechanism for alternative assembling is the replacement of constitutive ß1, ß2, and ß5 subunits with inducible ß1i, ß2i, and ß5i subunits, which confers a change in peptide substrate preference.^1,19 In addition, various endogenous post-translational modifications (PTMs) of proteasome subunits may also contribute to a heterogeneous assembly; the PTMs reported include phosphorylation,^23,24 N-terminal acetylation, and oxidation.²⁵ However, information regarding the modified amino acids on individual subunits, which is critical to our understanding of the structural arrangement and assembly of proteasome complexes, is scarce.

Characterization of proteasome complexes has been technically challenging because of the complexity in both protein structure and molecular composition. We have taken an organelle-targeted proteomic approach, which enabled us to overcome the difficulties and achieve a full characterization of the murine cardiac 26S proteasomes. This approach included (1) purification of 20S and 26S proteasomes by multidimensional liquid chromatography, (2) validation of purified intact proteasomes via an array of functional assays; (3) liquid chromatography/tandem mass spectroscopy (LC/MS/MS) based proteomic identification of molecular components expressed in this organelle; and (4) a final step of target validation by immunoblotting and immunocytochemistry to confirm identified proteins in cardiac myocytes. This comprehensive analysis of cardiac proteasomes has provided an essential proteomic blueprint of this important protein degradation machinery in the heart.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of the 20S and the 26S Proteasomes
The murine cardiac 20S proteasomes were purified by methodologies detailed by Zong et al²⁶ and are discussed in full in the online data supplement, available at available at http://circres.ahajournals.org. The 26S proteasomes were purified from 9-week-old male mice, wild-type ICR strain; a total of 1000 mice were used for this study. Experimental procedures (supplemental Figures I and II, available online) of the 26S proteasome purification are provided in the online data supplement.

Isolation of Adult Mouse Cardiomyocytes, Subcellular Fractionation, Electrophoresis, Immunoblotting, and Confocal Studies
Isolation of mouse cardiomyocytes and subcellular fractionation were performed as described in the online data supplement. One- or two-dimensional electrophoresis (1-DE or 2-DE), blue-native gel electrophoresis (BN-PAGE), immunoblotting, and confocal studies are also described in the online data supplement.

Liquid Chromatography/Tandem Mass Spectroscopy
26S Proteasomes were displayed by 1-DE, 2-DE, or BN-PAGE, and proteins were identified by LC/MS/MS (see the online data supplement). Product ion data were searched against the International Protein Index mouse protein database using a locally stored copy of the Sequest (Bioworks 3.2, Thermo) or Mascot search engine (Matrix Science). Search criteria included minimum Xcorr values of 2.0 for 1+, 2.2 for 2+, and 3.8 for 3+ ions (CN 0.1) for Sequest and a minimum Mascot score of 40 for each peptide. Furthermore, confirmation of a protein isoform was based on matching specific tryptic peptide fragments to a unique amino acid sequence of the isoform.²⁷

26S Proteasome Activity Assay
26S Proteasome activity was determined in ATP (2 mmol/L) containing HEPES buffer (50 mmol/L, pH 7.5) with the synthetic fluorogenic peptide substrate succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as detailed in the online data supplement.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The murine cardiac 26S proteasomes were isolated, and 26S proteasome–containing fractions were analyzed by immunoblotting (Figure 1A) and proteolytic activity assays (Figure 1D), confirming the expression of 19S subunits Rpt1 and Rpt4, as well as the 20S subunit 3, in glycerol fractions that exhibited high proteolytic activities (ie, fractions 30 to 32).

View larger version (17K): [in this window] [in a new window]   Figure 1. Purification and characterization of the murine cardiac proteasomes. A, Purification of the murine cardiac 26S proteasome by glycerol density gradient. Fractions collected from the glycerol gradient were analyzed by immunoblotting and proteasome activity assays. Immunoblotting showed that the 19S subunit Rpt4 and the 20S subunit 3 are present predominantly in glycerol fractions that exhibited high proteolytic activities, indicating the presence of high amounts of proteasome complexes in these fractions (ie, fractions 30 to 32). B, Blue-Native gel showing the existence of at least 2 species of murine cardiac 26S proteasomes, both of which were shown to contain 19S and 20S components by LC/MS/MS analyses. C, Confirmation of 20S, 19S, and 11S subunits in 2 26S species on a native gel by immunoblotting. The purified 26S proteasomes were run on a native gel and probed with antibodies to 3 (20S, left), Rpt4 (19S, center), PA28 (11S, right). D, Summary of proteolytic activities of the purified proteasome complexes. The 20S activity measured was the ß5 proteolytic activity. The 26S activity was measured in the presence of 2 mmol/L ATP without detergent.

Identification of Murine Cardiac 26S Proteasome Constitutive and Inducible Subunits
Using either 1-DE or 2-DE combined with LC/MS/MS, we achieved a full characterization of murine cardiac 26S proteasomes. A total of 36 unique subunits (19 for the 19S; 17 for the 20S) were identified, all (except one 19S subunit) with multiple unique peptides and high sequence coverage. Summaries of all identifications are shown in Tables 1 and 2. The integrity of purified 26S proteasomes was demonstrated by the BN-PAGE analysis (Figure 1B). Additional target validation by immunoblotting or LC/MS/MS of native-gel-displayed 26S proteasomes confirmed that there are at least 2 distinct species of 26S complexes in the heart, both of which expressed subunits from 20S and 19S proteasomes (Figure 1C). Furthermore, both purified 20S and 26S proteasomes exhibited strong proteolytic activities (Figure 1D). This is the first demonstration that there are multiple species of cardiac 26S proteasomes.

View this table: [in this window] [in a new window]   Table 1. LC/MS/MS Identified Murine Cardiac 20S Proteasome Subunits

View this table: [in this window] [in a new window]   Table 2. LC/MS/MS Identified Murine Cardiac 19S Proteasome ATPase and Non-ATPase Subunits

Characterization of Murine Cardiac 19S Proteasomes
Characterization of 19S proteasomes was most effective by LC/MS/MS analyses of 1-DE-displayed 19S proteasomes. The higher molecular mass of 19S subunits was more effectively separated by 1-DE (eg, Rpn1 and Rpn2 subunits; both >100 kDa). All identifications were made by multiple peptides with high sequence coverage, with the exception of S5b (6% sequence coverage); however, it was identified by 2 distinct matching peptides, and the data were reproducible. The molecular mass of 19S subunits ranged from 31 to 105 kDa (Table 2). Figure 2 illustrates an example of the identification of Rpn1, which is a large subunit (100.2 kDa) of 26S proteasomes. Twenty-nine distinct peptides were detected, resulting in sequence coverage of 53%.

View larger version (59K): [in this window] [in a new window]   Figure 2. Identification of the 19S subunit Rpn1 by LC/MS/MS. An example spectra of LC/MS/MS characterization of the proteasome subunits is given. Bolded amino acids represent peptides that were detected in Rpn1, which has a molecular mass of 100.2 kDa.

Examination of 11S and PA200
In addition to 19S complexes, 11S and PA200 are potential binding complexes of 20S proteasomes; they were found in yeast, liver, and skeletal muscle.^28,29 Two subunits of 11S, known as PA28 (subunit 1) and PA28ß (subunit 2), form a 180 to 200 kDa complex. Compared with liver, the heart expressed less 11S (Figure 3A). In the LC/MS/MS analyses of isolated 20S proteasomes, PA28 was found only once with 1 unique peptide and a total of 2 peptides (IPI00124225); PA28ß appeared once with 2 unique peptides and a total of 4 peptides (IPI00124223). The one-time detection of 11S subunits was from a total of 9 independently analyzed 20S proteasome samples (among them, 3 1-DE, 3 2-DE, and 3 BN-PAGE analyses coupled with LC/MS/MS). In addition, PA28 was found once as a single peptide in 1 26S sample from 4 independently analyzed 26S proteasome samples (2 1-DE, and 2 BN-PAGE analyses coupled with LC/MS/MS). Immunoblotting confirmed that PA28 was clearly associated with 20S proteasomes (Figure 3A), but was limited in purified 26S proteasomes (Figure 1C and 3A). The expression of PA200 was found in total heart lysates, but was not detected in any of the purified cytosolic 26S proteasome or 20S proteasome samples from the heart (Figure 3B), indicating that the functionality of purified cardiac proteasomes was independent of PA200.

View larger version (23K): [in this window] [in a new window]   Figure 3. Analysis of 11S and PA200. A, Detection of PA28 in 20S proteasomes by immunoblotting. Total heart homogenates, purified 26S proteasomes, and 20S proteasomes, were probed with anti-PA28 (left); (middle) cytosolic and nuclear fractions (15 µg each) probed with anti-PA28; (right) total heart, lung, and liver homogenates (15 µg each) probed with anti-PA28. B, Analysis of PA200 in the purified proteasomes. Total heart homogenates, purified 26S proteasomes, and 20S proteasomes, were probed with anti-PA200 (left). Cytosolic and nuclear fractions (30 µg each) probed with anti-PA200 (right). C, Relative amounts of different proteasome subunits in various murine tissues. Total tissue homogenates from murine brain, liver, kidney, lung, and heart samples as well as purified 20S proteasomes were probed with anti-7 (upper panel) or anti-Rpt4 (middle panel) and anti-ß2i (lower panel) without the 20S samples.

Characterization of Murine Cardiac 20S Inducible Subunits
All 3 inducible 20S subunits were detected in both purified 20S and 26S proteasomes (Table 1; Figure 3C). The expression of inducible subunits was previously shown in the liver by the stimulation of tumor necrosis factor or interferon .³⁰ However, the parallel detection of these inducible subunits together with their constitutive counterparts in the normal adult myocardium was unexpected. These inducible subunits were not previously detected in normal rat heart³¹ nor in human erythrocytes.³¹ Interestingly, the heart expressed relatively less 7 subunit (20S), less Rpt4 (19S), and less ß2i than that expressed in the liver, kidney, or lung (Figure 3C).

2-DE Map of 20S Proteasomes
The integrity of purified 20S proteasomes was further verified by their 2-DE map (Figure 4A), documenting all 14 constitutively expressed and ß subunits. More than 35 protein spots from each 2-DE were determined to be 20S subunits, indicating a high degree of complexity and heterogeneity of the cardiac 20S proteasomes compared with other cell types reported.^31–33 Our study represents one of the most comprehensive characterizations of the 20S proteasomes from any species. Table 1 lists all 20S subunits identified in the murine heart, reproducibly, from multiple 2-DE experiments. These subunits possess a wide range of isoelectric point (pI) (4.8 to 8.6) with molecular mass of 21 to 31 kDa.

View larger version (38K): [in this window] [in a new window]   Figure 4. 2-DE of purified murine cardiac 20S proteasomes. A, Experimental 2-DE (SYPRO Ruby stained) of purified murine cardiac 20S proteasomes displays all 14 constitutively expressed subunits as well as 1 inducible isoform ß5i. Subunit identifications were made by LC/MS/MS. The existence of multiple spots of a given subunit (eg, 3, 4, 5, 6, ß1, and ß7 isoforms) suggest PTMs. This experimental 2-DE was consistent with its theoretical 2-DE shown in B (generated with bioinformatic tools at www.expasy.org). C, Theoretical map of the yeast 20S proteasome subunits, which exhibits a clear distinction, with respect to molecular weight and pI values of the subunits, from that of the murine cardiac 20S proteasome 2-DE shown in panels A and B.

The experimental 2-DE map of purified 20S proteasomes (Figure 4A) was remarkably consistent with the theoretical murine 20S proteasome 2D map (Figure 4B), which was generated using theoretical subunit masses and pI values (www.expasy.org); the theoretical map did not consider PTMs. Importantly, the murine theoretical 20S map displayed distinct characteristics from that of the yeast (Figure 4C); differences are evident with respect to their subunit masses and pI values. Several murine cardiac 20S proteasome subunits (3, 4, 5, 6, ß1, ß7) were represented by multiple spots on the 2-DE, indicating PTMs of these subunits. It is conceivable that other subunits or associating partners were present on the experimental 2-DE but were not detected by SYPRO Ruby stain because they were in low abundance.

Confirmation of Cardiac Cell Origin and Subcellular Distribution
To determine whether proteasome complexes isolated from the murine heart were present in cardiomyocytes, murine cardiomyocytes were isolated. Immunoblotting (Figure 5A) and confocal microscopy (Figure 5B and 5C) documented the expression of proteasome subunits in cardiomyocytes. Importantly, all 3 inducible 20S subunits were detected in cardiomyocytes as well as in the purified cardiac 20S and 26S proteasomes (Figure 5A through 5C). The subcellular distributions of the 20S and the 19S proteasomes were examined. Confocal studies illustrated strong signal by antibodies against the 20S core subunits 5, 7, ß1, ß5, and ß7 in all subcellular fractions (Figure 5C), which is consistent with the immunoblotting analyses displaying high amounts of the 20S 3 subunit in the cytosol, endoplasmic reticulum/plasma membrane (ER/PM), and nuclear fractions (Figure 5D). The nuclear fraction displayed less signal of the 19S subunit Rpn2 (Figure 5B), as did the immunoblotting of Rpt4 (Figure 5D) in the nucleus compared with cytosol.

View larger version (56K): [in this window] [in a new window]   Figure 5. Verification of proteasome subunits expression in cardiomyocytes. A, Immunoblotting of proteasome subunits in myocytes, heart lysates, purified 20S proteasomes, and Hela cell lysate. B, Immunocytochemistry analyses of murine cardiomyocytes labeled with antibodies against Rpn2 (A) or estrogen receptor (ER) (B), and an overlay of Rpn2 and ER (C). Lower panels show higher resolution of the nuclear region. Green: Anti-ER, Red: Anti-Rpn2. C, Immunocytochemistry analyses of cardiomyocytes labeled with antibodies against core-20S (5, 7, ß1, ß5, ß7) or -actinin. Green: Anti–20S-core subunits, Red: Anti–-actinin. -Actinin was absent in nucleus. Lower panels show higher resolution of the nuclear region. D, Subcellular localization of 20S (upper panel, using anti-3 antibodies) and of 19S (lower panel, using anti-Rpt4 antibodies) proteasomes in the murine heart. Lane-1, Nucleus; Lane-2, ER/PM; Lane-3, mitochondria; Lane-4, cytosol. Lower panel: Lane-1, Cytosol; Lane-2, ER/PM; Lane-3, mitochondria; Lane-4, nucleus.

Identifications of Novel PTMs of 26S Subunits
The present study revealed 3 types of PTMs of proteasome subunits, including N-acetylation, N-myristoylation, and phosphorylation (Figure 6A through 6C; Tables 1 and 2). Phosphorylation of proteasome subunits is described elsewhere by our group.²⁶ We identified a novel myristoylation on the N-terminal of Rpt2 (Figure 6A). The nucleophilic carbonyl oxygen on the amide bond resulting from N-terminal myristoylation is clearly shown by the b1 ion, confirming that the modified N-terminal residue is glycine. Although the Rpt2 subunit has been previously found to contain N-terminal myristoylation in rice²¹ and yeast,³³ to our knowledge, this is the first demonstration that myristoylation of Rpt2, or any proteasome subunit, exists in mammalian cells. Importantly, myristoylation of Rpt2 subunit was consistently detected in all samples.

View larger version (56K): [in this window] [in a new window]   Figure 6. PTMs of 26S proteasome subunits. A, Mass spectra of the N-terminally myristoylated peptide from the 19S subunit Rpt2. The -amino group of glycine was modified (mass change of 210.2); B, Mass spectra of N-terminally acetylated peptide from the 20S subunit 7. The -amino group of serine was modified (mass change of 42). C, Mass spectra of N-terminally acetylated peptide from the 19S subunit Rpn6. The -amino group of alanine was modified (mass change of 42). D, Three alternatively-spliced mouse Rpn10 isoforms. Unique peptides by LC/MS/MS are highlighted. The alternatively spliced region detected is underlined. Three peptides contained the GER insertion (ASAAEAGIATPGTEGERDSDDALLK) whereas 3 other peptides did not (AAAASAAEAGIATPGTEDSDDALLK).

Ten proteasome subunits were N-terminally acetylated, including subunits Rpn1, Rpn5, Rpn6, Rpt3, Rpt6, 2, 5, 7, ß3, and ß4 (Tables 1 and 2). Acetylation of Rpn1 is a novel finding that, the best of our knowledge, has never been detected in humans, rice, or yeast. Examples of N-terminal acetylation of 7 and of Rpn6 are shown (Figure 6B and 6C). Collectively, the proteasome subunits have at least 2 types of N-terminal modifications (myristoylation and acetylation) or are free of any N-terminal modifications.

Alternatively Spliced 19S Subunit
The multi-ubiquitin chain-binding subunit Rpn10 (PSMD4, Swiss-Prot O35226), was found to occur in at least 2 isoforms in the purified cardiac 19S proteasomes (Figure 6D). The Rpn10b and Rpn10c isoforms are identical to Rpn10a, with the exception of a GER insertion at amino acid 254 (Figure 6D). Several peptides containing 3 amino acids (GER) and peptides missing these 3 amino acids were detected in the 26S samples. Because we did not detect any C-terminal peptides of the Rpn10c isoform, we attributed the alternatively spliced isoform in cardiac muscle as the Rpn10b isoform. It is known that Rpn10 transcripts exist in at least 5 distinct mRNA forms in mouse testis and embryonic stem cells.³⁴ However, the protein expression of these mRNAs has never been reported. Our data represents the first documentation of protein expression of an alternatively spliced Rpn10 isoform in any organism.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our study documents the first protein atlas of the cardiac 26S proteasomes, which serve as an essential protein degradation machinery in the heart. Taking an organelle proteomic approach, the murine cardiac proteasome subunits were characterized, including 19 subunits for the 19S complexes and 17 subunits for the 20S complexes. Furthermore, our analyses verified the expression of key subunits in cardiomyocytes and determined their subcellular distributions. Substantial analyses on both structure and function of the purified cardiac 20S proteasome was performed,²⁶ lending further credence to target validation of purified proteasomes. The cardiac 20S and 19S proteasomes exhibit unique features that are strikingly different from those reported in yeast, liver, or erythrocyte proteasomes.^31–33 Moreover, the expression of both 19S and 20S were less abundant in the murine heart compared with that in the murine liver, lung, or kidney. Finally, we have identified 3 distinct PTMs on proteasome subunits, including myristoylation, phosphorylation, and N-terminal acetylation, demonstrating high complexity in the molecular composition of proteasomes, suggesting potential mechanisms to regulate assembly and function of this organelle in the heart.

Molecular Composition of Murine Cardiac Proteasomes
To our surprise, in addition to the 14 constitutive subunits, all 3 known inducible ß subunits were detected in the 20S proteasomes of normal myocardium. These data suggest that the heart may exhibit an immunoactive phenotype, which is reminiscent of what was reported in the liver and spleen, but in contrast to erythrocytes where inducible subunits in normal cells were not reported.^31,35 A previous report found no inducible subunits in normal rat hearts.³⁶ The N-terminal peptides of ß1, ß2, and ß5i were found to be free of modification (Table 1). Processing of these subunits (as well as ß5, ß1i, and ß2i) results in an active Thr residue at the N-termini of each protein, which implicates the significance of their function.²⁰

11S and PA200 are known associating partners of proteasomes in yeast, liver, and skeletal muscle.²⁸ Among them, the 11S complex, which was found to be highly expressed in the liver as an activator complex of the 20S, was in lower abundance in the heart. The association of 11S with both purified 20S and 26S proteasomes was scarce (Figures 1C and 3A), suggesting that these complexes may contribute very little to cardiac cytosolic 26S assembly in the normal murine myocardium. Although PA200 was not detected in the intact 20S or 26S complexes (Figure 3B), it remains to be determined whether the free PA200 modulates the activity of the cardiac 20S and 26S proteasomes.

Among the 19S proteasomes, the ATPase complexes in the base of the lid belong to the AAA (ATPases associated with diverse cellular activities) superfamily and are responsible for the unfolding and translocation of substrates into the enclosed 20S proteolytic chamber. Importantly, BN-PAGE displayed 2 distinct species of fully assembled 26S proteasomes in the mouse myocardium (Figure 1B and 1C). It appears that the high molecular weight 26S complex may contain additional 19S particles compared with that of the lower-molecular-weight 26S complexes (Figure 1B) and, possibly, distinct assembly of the 20S complexes as well.

Characterization of cardiac 26S proteasomes demonstrates differences compared with yeast proteasomes. An example of this is the lack of detection of the putative mouse 19S proteasomal subunits PSMD9³⁷ and PSMD10 in the purified cardiac 26S samples. The PSMD9 was originally found to bind to 2 19S ATPase subunits.³⁷ Another previously proposed 19S subunit is gankyrin (PSMD10).³⁸ This protein was found to be highly expressed in human hepatocellular carcinomas,³⁸ but is not stably associated with 26S proteasomes.³⁸ The absence of gankyrin in cardiac 26S proteasomes is consistent with the notion that gankyrin is not a regular 26S subunit.

Database Conflicts for Putative Murine Proteasome Subunits
The Swiss-Prot database contains nonredundant and cross-referenced information on mouse proteasome subunits. Thus far, validation on sequence information of these proteasome subunits is limited. Our investigation provides the first comprehensive analysis of murine proteasome subunits and detailed information on their amino acid sequences. Specifically, our LC/MS/MS data resolve potential sequence conflicts reported in the predicted mouse proteasome amino acid sequence derived from their nucleotide sequences. For example, conflicts at residues 83 (HL), 214 (SF), and 222 (ML) of PSMD12 in the Swiss-Prot database (Q9D8W5) were found to be L83, F214, and L222, respectively, in the murine cardiac proteasome preparations. Conflicts in the Swiss-Prot database for Rpt1 (PSMC2, P46471) at residues 36 (GA), 38 to 39 (STRC), 121 to 122 (VVEE), 137 (MR), 187 (FL), and 276 (IM) were found to be G36, S38, T39, V121, V122, M137, F187, and I276, respectively, in the murine cardiac proteasomes. The amino acids identified in the mouse cardiac proteasomes at other conflicting residues in Rpt6 (PSMC5, P62196), R375, Rpt4 (PSMC6, P62334), E144, A222 and ß2 (PSMB7, P70195), K237 were also determined. The 19S proteasome subunit S5b (PSMD5), a subunit with virtually undefined function, was identified reproducibly in the mouse heart and was found in human red blood cell 26S proteasome preparations.³⁹

PTMs on Cardiac Proteasome Subunits
N-terminal myristoylation, N-terminal acetylation, and phosphorylation of cardiac proteasome subunits were observed. N-terminal protein myristoylation has been shown to regulate protein targeting and function by promoting weak and reversible protein–membrane and protein–protein interactions.^40,41 Rpt2 was N-terminally myristoylated; although the functional significance of this remains to be determined, it may be important in targeting the 26S proteasomes to membranes. N-terminal acetylation of protein affects the stability, function, and degradation of proteins.⁴² N-terminally acetylated subunits were found in 10 proteasome subunits. Interestingly, we observed N-terminal acetylation of Rpn1 in heart, whereas Rpn1 in yeast is not N-terminally acetylated.³³ The mammalian Rpn1 has not been previously shown to be acetylated. In contrast, the 20S subunits have been found to be acetylated in both species. Additional studies on characterization of 20S subunits phosphorylation was conducted; an important regulatory role of phosphorylation in 20S proteolytic function in the heart was established.²⁶

Novel Alternatively Spliced 19S Subunits
Protein sequences of alternatively spliced subunit isoforms have not been previously reported. The transcript variants of Rpn10 (PSMD4), which appear to have been generated by a single gene, were shown in at least 5 distinct mRNA forms (Rpn10a-10e) in mouse testis and embryonic stem cells.³⁴ In our study, distinct peptides of the alternatively spliced region confirmed the existence of 2 Rpn10 isoforms in the mouse heart. Based on the distinct Rpn10 peptides identified, the most likely Rpn10 isoforms present in the cardiac proteasome are Rpn10a and Rpn10b (Figure 6D). Given the significance of Rpn10 as a major polyubiquitin binding subunit of the 19S complexes, the existence of 2 alternative spliced Rpn10 isoforms in the heart may have tremendous functional implications.

Conclusion
Recent studies have implicated proteasome dysfunction in myocardial ischemic injury. Because some investigations have yielded conflicting results, they generated the debate of whether pharmacological inhibition of proteasome function is therapeutically beneficial for cardiac diseases. The full characterization of murine cardiac 26S proteasomes provides essential information that is fundamental to our understanding of the function and regulation of this organelle. The demonstration of multiple species of 26S proteasomes in the heart suggests the possibility of subpopulation-dependent function and regulation, which may be critical to future investigations on elucidating proteasome function in cardiac disease phenotypes.

	Acknowledgments

 
Sources of Funding

This work was supported by NIH grants HL63901, HL65431, and HL080111 to P.P., an American Heart Association Predoctoral Fellowship (0315086B) to C.Z., and an endowment from Theodore C. Laubisch to P.P.

Disclosures

None.

	Footnotes

 
Original received February 25, 2006; revision received June 12, 2006; accepted July 7, 2006.

	References

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