Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3

Objective: Regulation of proteasomal activity is an essential component of cellular proteostasis and function. This is evident in patients with mutations in proteasome subunits and regulators, who suffer from proteasome-associated autoinflammatory syndromes (PRAAS). These patients display lipodystrophy and fevers, which may be partly related to adipocyte malfunction and abnormal thermogenesis in adipose tissue. However, the cell-intrinsic pathways that could underlie these symptoms are unclear. Here, we investigate the impact of two proteasome subunits implicated in PRAAS, Psmb4 and Psmb8, on differentiation, function and proteostasis of brown adipocytes. Methods: In immortalized mouse brown pre-adipocytes, levels of Psmb4, Psmb8, and downstream effectors genes were downregulated through reverse transfection with siRNA. Adipocytes were differentiated and analyzed with various assays of adipogenesis, lipogenesis, lipolysis, inflammation, and respiration. Results: Loss of Psmb4, but not Psmb8, disrupted proteostasis and adipogenesis. Proteasome function was reduced upon Psmb4 loss, but partly recovered by the activation of Nuclear factor, erythroid-2, like-1 (Nfe2l1). In addition, cells displayed higher levels of surrogate inflammation and stress markers, including Activating transcription factor-3 (Atf3). Simultaneous silencing of Psmb4 and Atf3 lowered inflammation and restored adipogenesis. Conclusions: Our study shows that Psmb4 is required for adipocyte development and function in cultured adipocytes. These results imply that in humans with PSMB4 mutations, PRAAS-associated lipodystrophy is partly caused by disturbed adipogenesis. While we uncover a role for Nfe2l1 in the maintenance of proteostasis under these conditions, Atf3 is a key effector of inflammation and blocking adipogenesis. In conclusion, our work highlights how proteasome dysfunction is sensed and mitigated by the integrated stress response in adipocytes with potential relevance for PRAAS patients and beyond.

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria

Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each other's functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads

Constitutive- and immunoproteasomes are part of the ubiquitin–proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the β5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and β5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the β5, β5i, β1, and β1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either β5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

High-Dose Carfilzomib Recaptures Response in Relapsed/Refractory Multiple Myeloma Resistant to Low-Dose Carfilzomib By Co-Inhibiting β2 Subunit of Proteasome Complex: The First in Human Evidence

Background Proteasome, a complex involved in the intracellular protein degradation, consists of multiple subunits, but only three subunits have enzymatic activity to cleave and degrade proteins, namely β1, β2 and β5. Carfilzomib (CFZ), a second-generation proteasome inhibitor (PI), can induce cell death by selective and irreversible inhibition of β5 subunit of proteasome. Preclinical data suggested that high-dose CFZ could co-inhibit predominantly β2 proteasome activity, followed by β1 inhibition (Besse et al, Cell Chem Biol. 2019). Over the past few years, CFZ has become a corner stone for multiple myeloma (MM) therapy. Currently, CFZ is approved by the FDA in different dosing schedules in combination with lenalidomide or daratumumab and dexamethasone. However, the optimal CFZ dosing is still a matter of debate, with the approved dosage ranging from 20to 70mg/m 2 in different regimens. In addition, if response can be recaptured by escalating CFZ dose in patients progressing from low-dose CFZ has yet to be determined. The aim of our current study was to analyse the profile of proteasome inhibition in the respective dose cohorts and to elucidate if high-dose CFZ could recapture response in patients resistant to low-dose CFZ. Methods We prospectively collected clinical data and peripheral blood mononuclear cells (PBMC) of 32 patients with relapsed/refractory (RR) MM before and 1-8 hours after CFZ administration. PBMC were lysed and labelled for the activity of individual proteasome subunits using activity based proteasome probes and the proteasome subunits were separated using SDS-PAGE. The activity of constitutive and immunoproteasome β1, β2 and β5 subunits was evaluated by densitometry analysis and combination of the activity of constitutive and immunoproteasome individual subunit was used for further analysis. Results Overall, six, nine, twelve and five patients received CFZ at a dose of 20, 27, 36 and 56 mg/m 2, respectively. As expected, the total activity of proteasome decreased with higher doses of CFZ. Significant inhibition (median inhibition > 50%) of β5 subunit was observed already at 20 mg/m 2 dose, while β2 subunit started to be co-inhibited only at a dose of ≥27 mg/m 2. Significant co-inhibition of β2 activity was seen at 36 mg/m 2 dose, at which also β1 subunit started to be co-inhibited. Finally, at 56 mg/m 2, the activity of all active subunits was inhibited with a median inhibition of > 50%, with the strongest inhibition of the β5 subunit, followed by β2 and then β1. When we compared the patient groups low-dose CFZ (20 or 27 mg/m 2) versus high-dose CFZ (36 or 56 mg/m 2), we observed a significant difference in β2 (P=0.002) and β5 (P=0.02) subunit inhibition between the both groups. In terms of total proteasome activity, high-dose CFZ demonstrated a significantly higher proteasome inhibition in comparison with patients receiving low-dose CFZ (P=0.01). In brief, our results suggested that high-dose CFZ, in contrast to low-dose CFZ, could obtain superior proteasome inhibition by co-inhibiting β2 subunit of proteasome complex. In light of this finding, we successfully treated six RRMM patients who were resistant to low-dose CFZ with CFZ dose escalation. All six patients were heavily pretreated with 3-12 lines of therapy including daratumumab, two PIs, two immunomodulatory drugs and autologous stem cell transplant. Additionally, one and two patients received prior treatment with B-cell maturation antigen targeted bi-specific antibody and chimeric antigen receptor modified T-cell, respectively. In the last line of treatment, these six patients showed progression during CFZ based regimens with low-dose CFZ, namely 20 or 27 mg/m 2. We therefore increased the CFZ dose to 36 or 56 mg/m 2 and the doses of agents other than CFZ in the combination regimens remained the same. High-dose CFZ dose recaptured response in all six patients with four and two patients that achieved partial remission and very good partial remission, respectively, and the progression free survival ranged from 1-13 months. Conclusion In summary, high-dose CFZ, namely ≥ 36mg/m 2, showed more effective proteasome inhibition via blocking β5 and β2 subunits, while low-dose CFZ could not achieve a sufficient inhibition of β2 subunit. We provided the first in human evidence that high-dose CFZ could recapture response in RRMM patients resistant to low-dose CFZ by co-inhibiting the β2 subunit activity of proteasome complex. Figure 1 Figure 1. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

PSMB4 and PSMD4 Are Correlated with 1q21 Amplification in CD138 + Plasma Cells: New Potential Druggable Targets in Myeloma Patients

The amplification of the 1q21 (amp1q21) region is one of the most acquired cytogenetic abnormalities (CA) in multiple myeloma (MM) associated with a worse patient outcome and disease progression. Moreover, different studies have demonstrated that the number of copies (CN) 1q21 (gain1q21: three copies or amp1q21: ≥ four copies) have a different impact in the response to anti-MM therapies. Particularly, it has been proposed that in MM patients, additional copies of 1q21 may be associated with the resistance to proteasome inhibitor (PI) treatment as bortezomib. A recent study showed that newly diagnosed MM (MMD) patients carrying amp1q21 but not gain1q21 receiving carfilzomib-based treatment have an early disease progression with shorter overall survival. Previous studies underlined that the amplification of 1q21 can lead to the overexpression and/or dysregulation of several candidate genes associated with cell proliferation, apoptosis, and drug resistance. Here we aim to identify 1q21 target genes possibly correlated to the response to PI therapy. We evaluated a total cohort of 29 primary plasma cells (PCs) purified from bone marrow (BM) blood aspirates from 11 smoldering MM (SMM) and 18 MMD. The median age of our cohort was 70 years (range: 38-86). Fluorescence in situ hybridization (FISH) analysis was performed to access the presence or absence of copy number alteration (CNA) in the 1q21 region in all patients. 14 out of 29 patients carried 1q21 CNA (5 with gain1q21 and 9 with amp1q21). A score reflecting the number of 1q21 copies was calculated based on the hybridization pattern. The transcriptional profiles of the 29 BM PCs samples were generated on GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The samr package was used in R for call genes as differentially expressed between 1q21 CN-altered and wild-type samples. The correlation between the 1q21 copy number score and the gene expression levels was performed. Moreover, we have evaluated by FISH the 1q21 CNA in a panel of human myeloma cell lines (HMCLs): OCY-MY5, JJN3, RPMI-8226, NCI-H929, and OPM2. JJN3 were transfected with a control vector and PSMB4 and PSMD4 short hairpin RNA (shRNA) lentivectors. The gene and protein expression levels of PSMB4 and PSMD4 in MM cell lines were analyzed by qRT-PCR and Western Blot, respectively. Cell viability and proliferation were assessed using MTT assay and flow cytometry. Our bioinformatic analyses highlighted the overexpression of different genes (IL6R, ILF2, BCL9, MCL1, CSk1B, ADAR1, ARNT, ANP32E) in the 1q21 CNA samples with respect to the controls, as already reported in the literature. Our analysis showed a significantly higher expression of two proteasome subunits (PSMB4 and PSMD4) in patients with 1q21 CNA when compared with patients without (PSMB4 p=0.0006; PSMD4 p=<0.0001). Patients with amp1q21 showed a higher expression of PSMB4 when compared to the patients with gain1q21 (p=0,007). In our cohort, gene expression profile analysis also showed a strong positive correlation between gene expression levels and 1q21 CN for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, we found that the PSMB4 and PSMD4 expression level was independent of the disease stage (SMM vs MM) and was only driven by 1q21 CN. We have evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type cell line (OCY-MY5) and in a panel of MM cell lines carrying different degrees of 1q21 CN (in order: JJN3, U266, RPMI-8226, OPM2, and NCI-H929). The mRNA expression level of PSMB4 and PSMD4 was higher in cell lines carrying 1q21 amp, following a 1q21 copy number fashion. Similar results were obtained when protein levels of MM cell lines were analyzed by Western Blot. To further determine the potential role of both proteasome subunits in the pathogenesis of amp1q21, we generated a PSMB4-shRNA and PSMD4-shRNA knockdown stable MM cell lines. Functional studies showed that blockade of PSMB4 and PSMD4 decreased MM cell viability. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying amp1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with ampq21 Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Bristol Mayers Squibb: Other: congress; GSK: Other: clinical studies; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies.

Multi-omic analysis reveals the biochemical changes underpinning the varied phenotypes of the Arabidopsis long-period mutant rve 4 6 8

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we demonstrate how RVE8-like proteins impact diel plant cell function and draw novel connections to a number of plant cell processes that underpin the growth and development phenotypes observed in rve 4 6 8 plants. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at ZT0. We further demonstrate that RVE8-like proteins have a unique impact on the abundance and phosphorylation of 26S proteasome subunits, in addition to impacting the abundance and phosphorylation status of a number of protein kinases. Overall, this robust, multi-omic dataset, provides substantial new insights into RVE8-like protein function and the far reaching impact RVE8-like proteins have on the diel plant cell environment.

Neurodevelopmental Disorders (NDD) Caused by Genomic Alterations of the Ubiquitin-Proteasome System (UPS): the Possible Contribution of Immune Dysregulation to Disease Pathogenesis

Over thirty years have passed since the first description of ubiquitin-positive structures in the brain of patients suffering from Alzheimer’s disease. Meanwhile, the intracellular accumulation of ubiquitin-modified insoluble protein aggregates has become an indisputable hallmark of neurodegeneration. However, the role of ubiquitin and a fortiori the ubiquitin-proteasome system (UPS) in the pathogenesis of neurodevelopmental disorders (NDD) is much less described. In this article, we review all reported monogenic forms of NDD caused by lesions in genes coding for any component of the UPS including ubiquitin-activating (E1), -conjugating (E2) enzymes, ubiquitin ligases (E3), ubiquitin hydrolases, and ubiquitin-like modifiers as well as proteasome subunits. Strikingly, our analysis revealed that a vast majority of these proteins have a described function in the negative regulation of the innate immune response. In this work, we hypothesize a possible involvement of autoinflammation in NDD pathogenesis. Herein, we discuss the parallels between immune dysregulation and neurodevelopment with the aim at improving our understanding the biology of NDD and providing knowledge required for the design of novel therapeutic strategies.

α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast

Parkinson’s disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus niger in Peanuts

Aspergillus niger is a very destructive pathogen causing severe peanut root rot, especially in the seeding stage of peanuts (Arachis hypogaea), and often leading to the death of the plant. Protein lysine 2-hydroxyisobutyrylation (Khib) is a newly detected post-translational modification identified in several species. In this study, we identified 5041 Khib sites on 1,453 modified proteins in A. niger. Compared with five other species, A. niger has conserved and novel proteins. Bioinformatics analysis showed that Khib proteins are widely distributed in A. niger and are involved in many biological processes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that Khib proteins were significantly enriched in many cellular compartments and pathways, such as ribosomes and proteasome subunits. A total of 223 Khib proteins were part of the PPI network, thus, suggesting that Khib proteins are associated with a large range of protein interactions and diverse pathways in the life processes of A. niger. Several identified proteins are involved in pathogenesis regulation. Our research provides the first comprehensive report of Khib and an extensive database for potential functional studies on Khib proteins in this economically important fungus.