Proteasome Complex Research Articles

Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

AbstractDeubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn2+ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn2+ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.

Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes.

SummaryProteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

Cellular Responses to Proteasome Inhibition: Molecular Mechanisms and Beyond.

Proteasome inhibitors have been actively tested as potential anticancer drugs and in the treatment of inflammatory and autoimmune diseases. Unfortunately, cells adapt to survive in the presence of proteasome inhibitors activating a variety of cell responses that explain why these therapies have not fulfilled their expected results. In addition, all proteasome inhibitors tested and approved by the FDA have caused a variety of side effects in humans. Here, we describe the different types of proteasome complexes found within cells and the variety of regulators proteins that can modulate their activities, including those that are upregulated in the context of inflammatory processes. We also summarize the adaptive cellular responses activated during proteasome inhibition with special emphasis on the activation of the Autophagic-Lysosomal Pathway (ALP), proteaphagy, p62/SQSTM1 enriched-inclusion bodies, and proteasome biogenesis dependent on Nrf1 and Nrf2 transcription factors. Moreover, we discuss the role of IRE1 and PERK sensors in ALP activation during ER stress and the involvement of two deubiquitinases, Rpn11 and USP14, in these processes. Finally, we discuss the aspects that should be currently considered in the development of novel strategies that use proteasome activity as a therapeutic target for the treatment of human diseases.

Effects of first-generation in utero exposure to diesel engine exhaust on second-generation placental function, fatty acid profiles and foetal metabolism in rabbits: preliminary results

Atmospheric pollution has major health effects on directly exposed subjects but intergenerational consequences are poorly characterized. We previously reported that diesel engine exhaust (DE) could lead to structural changes in the placenta of in utero exposed rabbits (first generation, F1). The effects of maternal exposure to DE were further studied on second-generation (F2) rabbits. Pregnant F0 females were exposed to filtered, diluted DE (1 mg/m3, median particle diameter: 69 nm) or clean filtered air (controls) for 2 h/day, 5 days/week by nose-only exposure during days 3–27 post-conception (dpc). Adult female offspring (F1) were mated to control males: F1 tissues and F2 foeto-placental units were collected at 28 dpc and placental structure and gene expression (microarray) analysed. Fatty acid profiles were determined in foetal and maternal plasma, maternal liver and placenta. In F1, compared to controls, hepatic neutral lipid contents were increased in exposed animals without change in the blood biochemistry. In F2, the placental lipid contents were higher, with higher monounsaturated fatty acids and reduced pro-inflammatory arachidonic acid (AA), without placental structural changes. Conversely, the proportion of anti-inflammatory n-3 polyunsaturated fatty acids in F2 plasma was increased while that of AA was decreased. Gene set enrichment analyses (GSEA) of F2 placenta transcriptomic data identified that the proteasome complex and ubiquitin pathways genes were over-represented and ion channel function and inflammation pathways genes were under-represented in exposed animals. These preliminary results demonstrate that diesel engine exhaust exposure and in utero indirect exposure should be considered as a programming factor within the context of the DOHaD (Developmental Origins of Health and Disease) with a probable intergenerational transmission.

Reverse immunology: From peptide sequence to tumor-killing human T-cell clones.

Recent advances in next generation sequencing expanded the availability of tumor mutanome data that list the mutations present in cancer cells. Mutated proteins are an interesting source of neoantigens that can be used to specifically target tumor cells in the context of immunotherapy. However, identifying new antigenic peptides from mutated proteins remains challenging. In this chapter, we present Reverse Immunology as an approach to identify potential antigens from any given polypeptide sequence. First, we explain the rationale behind the identification of candidate HLA-binding peptides through mass spectrometry or in silico approaches. Then, we describe the isolation of low-frequency T-cell precursors specific for the candidate peptides using peptide-HLA multimers. Finally, we discuss validation steps leading to the identification of a T-cell clone recognizing tumor cells that endogenously process the candidate peptide. We also present approaches to study the impact of the proteasome complex on candidate peptide processing.

Expressions and characterization of MuRFs, Atrogin-1, F-box25 genes in tilapia, Oreochromis niloticus, in response to starvation.

Muscle accretion is affected by the difference between protein synthesis and its degradation. Studies on different species revealed that muscle proteolysis is mediated by different pathways including the ubiquitin-proteasome pathway in which the ubiquitin protein ligases play an important role. These muscle atrophy associated ligases were not well studied in tilapia. In this study, we characterized the ubiquitin protein ligases MuRF1/2/3, Atrogin-1 and F-box25, members of the ubiquitin-proteasome pathway in tilapia, Oreochromis niloticus, and their expressions in the muscle of starved, fed, refed, and control fish. Sequences of these genes revealed presence of Ring finger, B-box, and Cos domains in all MuRF genes, as well as F-box domain in Atrogin-1 and F-box25 genes. Real-time qPCR data analysis showed that expression of MuRF1/2/3, Atrogin-1, F-box25, and proteasome complex genes was significantly upregulated in starved fish compared to fed fish. Concurrently, the proteasome activity was 1.7-folds elevated in the starved fish compared to fed fish. These results confirm the important role of these genes in muscle degradation and suggest potential usage as markers of muscle accretion in tilapia.

Pharmacodynamics and pharmacokinetics of proteasome inhibitors for the treatment of multiple myeloma

ABSTRACTIntroduction: Multiple myeloma (MM) is the second most commonly diagnosed hematologic malignancy and has an increasing incidence and prevalence globally, and proteasome inhibitors (PIs) form the backbone of some of our most effective regimens for all phases of this disease in fit and frail patients.Areas covered: Strong understanding of the proteasome complex is increasingly important as the rapid development of new PIs and innovative myeloma therapies complicate the use of old and new combination regimens. We focus herein on the pharmacodynamics and pharmacokinetics of the approved PIs and others in development, including their safety and efficacy in corresponding clinical studies.Expert opinion: Advancements such as the first oral PI, ixazomib, with a more convenient route of administration and improved toxicity profile led to an improved quality of life, patient compliance, and all-oral combination regimens which are efficacious for long-term management of standard and high-risk MM. Novel pan-PIs, such as marizomib, hold the promise of superior clinical activity due to irreversible targeting of all multicatalytic proteinase complex subunits. Development of clinically validated biomarkers of PI sensitivity/resistance is required to inform utilization of the most optimal and effective, rationally targeted PI treatments for all MM patients.

Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.

Protein synthesis inhibition induces proteasome assembly and function

Protein synthesis and degradation balance have a crucial role in maintenance of cellular homeostasis and function. The ubiquitin-proteasome system is one of the major cellular proteolytic machineries responsible for the removal of normal, abnormal, denatured or in general damaged proteins. Proteasome is a multisubunit enzyme that consists of the 20S core and the 19S regulatory complexes giving rise to multiple active forms. In the present study we investigated the crosstalk between protein synthesis and proteasome-mediated protein degradation. Pharmacological protein synthesis inhibition led to increased proteasome function and assembly of 30S/26S proteasome complexes, in human primary embryonic fibroblasts. The enhancement in proteasome function counted for the degradation of ubiquitinated, misfolded and oxidized proteins. Additionally, it was found that heat shock proteins 70 and 90 are probably involved in the elevated proteasome assembly. Our results provide an insight on how the mechanisms of protein synthesis, protein degradation and heat shock protein chaperones machinery interact under various cellular conditions.

Altered proteasome function in right ventricular hypertrophy

In patients with pulmonary hypertension, right ventricular hypertrophy (RVH) is a detrimental condition that ultimately results in right heart failure and death. The ubiquitin proteasome system has been identified as a major protein degradation system to regulate cardiac remodelling in the left heart. Its role in right heart hypertrophy, however, is still ambiguous. RVH was induced in mice by pulmonary artery banding (PAB). Both, expression and activity of the proteasome was found to be up-regulated in the hypertrophied right ventricle (RV) compared to healthy controls. Catalytic inhibition of the proteasome by the two proteasome inhibitors Bortezomib (BTZ) and ONX-0912 partially improved RVH both in preventive and therapeutic applications. Native gel analysis revealed that specifically the 26S proteasome complexes were activated in experimental RVH. Increased assembly of 26S proteasomes was accompanied by elevated expression of Rpn6, a rate-limiting subunit of 26S proteasome assembly, in hypertrophied cardiomyocytes of the right heart. Intriguingly, patients with RVH also showed increased expression of Rpn6 in hypertrophied cardiomyocytes of the RV as identified by immunohistochemical staining. Our data demonstrate that alterations in expression and activity of proteasomal subunits play a critical role in the development of RVH. Moreover, this study provides an improved understanding on the selective activation of the 26S proteasome in RVH that might be driven by the rate-limiting subunit Rpn6. In RVH, Rpn6 therefore represents a more specific target to interfere with proteasome function than the commonly used catalytic proteasome inhibitors.

Comparative Analysis of Proteins in Extracellular Vesicles from Quiescent versus Activated Hepatic Stellate Cells

AimsA newly recognized mechanism of cell communication is intercellular trafficking of molecules that are contained within nano‐sized extracellular vesicles (EVs) such as exosomes or microvesicles. EVs mediate cell‐cell transfer of their cargo (miRs, mRNAs, proteins) resulting in functional reprograming of recipient cells. Hepatic stellate cells (HSC) are a normally‐quiescent cell type in the liver but after hepatic injury they transdifferentiate into collagen‐producing myofibroblasts that account for the majority of the fibrotic scar that is deposited during chronic liver injury. Recent studies have shown that EVs from quiescent HSC have anti‐fibrogenic properties whereas those from activated HSC are pro‐fibrogenic. To help understand these distinct properties, proteins in EVs from quiescent versus activated HSC were evaluated by mass spectrometry.MethodsNanoparticle tracking and Western blot confirmed the presence of EVs after differential ultracentrifugation of serum‐free conditioned medium from quiescent (Day 1–4) or activated (passage 1 (P1)) primary mouse HS. Five separate D4 EV preparations and three separate P1 EV preparations were individually subjected to mass spectrometry. Data were analyzed using Mascot (Matrix Science, London, UK) while MS/MS based peptide and protein identifications were validated with Scaffold (Proteome Software Inc., Portland, OR). Peptide identification was accepted if there was > 95% probability of FDR < 1.0% by the Peptide Prophet algorithm with Scaffold delta‐mass correction. Protein identifications were accepted if there was >99.0% probability of FDR < 1.0% with at least 2 constituent peptides identified. Protein probabilities were assigned by the Protein Prophet algorithm.Results46 proteins were identified in at least three of five D4 EV samples while 337 proteins were identified in three P1 EV samples. D4 EVs contained 19 specific proteins while P1 EVs contained 310 specific proteins. There were 27 proteins that were shared between both groups, with 11 of these proteins present at significantly higher levels in D4 EVs compared to P1 EVs. EVs from both groups contained exosomal proteins, but D4 EVs exhibited higher abundance of nucleosome components (histones, 17.4%, 46.7‐fold enrichment) or keratin filaments (15.2%, 57.6‐fold enrichment) while P1 EVs contained more proteins associated with extracellular space (30.9%, 4.0‐fold), proteasome complex (6.8%, 24.3‐fold), basement membrane (6.5%, 14.5‐fold), and collagen trimer (3.9%, 10.0‐fold). KEGG analysis showed 1 unique pathway for D4 EVs, 33 unique pathways for P1 EVs and 4 shared pathways for D4 and P1 EVs.ConclusionsEVs produced by activated HSC contain a protein cargo that is qualitatively and quantitatively different from that of EVs from quiescent HSC. These differences may account for distinct biological responses when each EV population acts on various hepatic cells, thus contributing to their differential pathophysiological or therapeutic outcomes. RC and XL contributed equally to this work.Support or Funding InformationNIH AA023626 & AA025974This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interplay between the Endogenous Opioid System and Proteasome Complex: Beyond Signaling.

Intracellular signaling mechanisms underlying the opioid system regulation of nociception, neurotransmitters release, stress responses, depression, and the modulation of reward circuitry have been investigated from different points of view. The presence of the ubiquitin proteasome system (UPS) in the synaptic terminations suggest a potential role of ubiquitin-dependent mechanisms in the control of the membrane occupancy by G protein-coupled receptors (GPCRs), including those belonging to the opioid family. In this review, we focused our attention on the role played by the ubiquitination processes and by UPS in the modulation of opioid receptor signaling and in pathological conditions involving the endogenous opioid system. The collective evidence here reported highlights the potential usefulness of proteasome inhibitors in neuropathic pain, addictive behavior, and analgesia since these molecules can reduce pain behavioral signs, heroin self-administration, and the development of morphine analgesic tolerance. Moreover, the complex mechanisms involved in the effects induced by opioid agonists binding to their receptors include the ubiquitination process as a post-translational modification which plays a relevant role in receptor trafficking and degradation. Hence, UPS modulation may offer novel opportunities to control the balance between therapeutic versus adverse effects evoked by opioid receptor activation, thus, representing a promising druggable target.

Proteasome \u03b25 subunit overexpression improves proteostasis during aging and extends lifespan in Drosophila melanogaster

The β5 subunit of the proteasome has been shown in worms and in human cell lines to be regulatory. In these models, β5 overexpression results in upregulation of the entire proteasome complex which is sufficient to increase proteotoxic stress resistance, improve metabolic parameters, and increase longevity. However, fundamental questions remain unanswered, including the temporal requirements for β5 overexpression and whether β5 overexpression can extend lifespan in other species. To determine if adult-only overexpression of the β5 subunit can increase proteasome activity in a different model, we characterized phenotypes associated with β5 overexpression in Drosophila melanogaster adults. We find that adult-only overexpression of the β5 subunit does not result in transcriptional upregulation of the other subunits of the proteasome as they do in nematodes and human cell culture. Despite this lack of a regulatory role, boosting β5 expression increases the chymotrypsin-like activity associated with the proteasome, reduces both the size and number of ubiquitinated protein aggregates in aged flies, and increases longevity. Surprisingly, these phenotypes were not associated with increased resistance to acute proteotoxic insults or improved metabolic parameters.

Deregulation of the 19S proteasome complex increases yeast resistance to 4-NQO and oxidative stress via upregulation of Rpn4- and proteasome-dependent stress responsive genes.

The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.

Expanding the Clinical and Molecular Spectrum of PSMD12-Related Neurodevelopmental Syndrome: An Additional Patient and Review

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process that eukaryotic cells use to regulate their functions and coordinate their signaling networks. In particular, it plays a crucial role in neuronal development regulating very important functional and morphological interplays of neurons, such as synaptic plasticity, neurotransmitter release and morphogenesis of axons. Pathogenic alterations of genes involved in the proteolysis by UPS have been associated with several neurodevelopmental disorders (NDDs) and human diseases, highlighting the importance of this regulatory mechanism to developmental processes and neurogenesis. Here, we describe a 19 years old male patient showing a syndromic form of NDD. The main clinical features are intellectual disability/speech delay, congenital anomalies and facial dysmorphisms. Through a targeted resequencing approach (TRS), we identified a missense variant in PSMD12, a gene recently associated to an emerging syndromic form of NDD, which encodes for the non-ATPase subunit of the 19S regulator of 26S proteasome complex. The variant described herein, inherited from the father with apparently borderline cognitive ability, is useful to expand the molecular spectrum of heterozygous PSMD12 mutations and to provide insight into the molecular pathogenesis of this new condition since it is, to the best of our knowledge, the first missense substitution to date reported in medical literature. More importantly, our study highlight once again the utility of next generation sequencing in establishing an etiological basis in clinically and genetically heterogeneous conditions such as NDDs, thus allowing a better diagnosis, counseling and management of affected patients and their families.

N-terminal HCV core protein fragment decreases 20S proteasome activity in the presence of PA28γ

The Hepatitis C virus (HCV) core protein plays a crucial role in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Its involvement in these diseases is reportedly abolished by a knockout of the proteasome activator PA28γ gene in transgenic mice, suggesting an interaction between the core protein and the PA28γ-proteasome system. This study found a direct interaction between the N-terminal 1–71 fragment of HCV core protein (Core71) and PA28γ in vitro, and that this interaction was found to enhance PA28γ-20S proteasome complex formation. While 20S proteasome activity was increased by PA28γ, it was significantly reduced by Core71 attachment in a dose-dependent manner. These results suggest that the Core-PA28γ interaction has an important role in regulating 20S proteasome activity and furthers our understanding of the pathogenesis of HCV.

Proteomic Signature of Neuroblastoma Cells UKF-NB-4 Reveals Key Role of Lysosomal Sequestration and the Proteasome Complex in Acquiring Chemoresistance to Cisplatin.

Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analyzed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analyzed using nano liquid chromatography with tandem mass spectrometry. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ATP-binding cassette superfamily proteins (ATP = adenosine triphosphate), solute carrier-mediated trans-membrane transporters, proteasome complex subunits, and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of the key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.

A SUMO ligase AtMMS21 regulates activity of the 26S proteasome in root development

The 26S proteasome is a multi-subunit protease controlling most of the cytosolic and nuclear protein turnover, regulating many cellular events in eukaryotes. However, functional modification on this complex remains unclear. Here, we showed a novel mechanism that a SUMO ligase AtMMS21 regulates activity of the 26S proteasome in root development of Arabidopsis. Our in vitro and in vivo data supported that AtMMS21 interacts with RPT2a, a subunit of the 26S proteasome. The mutants of AtMMS21 and RPT2a display similar developmental defect of roots, suggesting their association in this process. In addition, RPT2a is modified by SUMO3, potentially related to AtMMS21. During development, the activity of the 26S proteasome is lower in both mutants of AtMMS21 and RPT2a, compared with that of wild type. Furthermore, the protein level but not the RNA level of RPT2a is decreased in the absence of AtMMS21, implying stability regulation of the proteasome complex through the AtMMS21-RPT2a interaction. Taken together, the current study would improve our understanding on the regulatory mechanism of the 26S proteasome via protein modification in root development.