Protein Degradation Machinery Research Articles

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis.

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

Inhibition of 19S proteasome deubiquitinating activity in Schistosoma mansoni affects viability, oviposition, and structural changes.

The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.

Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry.

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Significance of Selective Protein Degradation in the Development of Novel Targeted Drugs and Its Implications in Cancer Therapy

AbstractThe evolution of traditional chemotherapeutic drugs to targeted drugs has led to major changes in cancer treatment. However, it remains difficult to develop effective targeted drugs that can act against “undruggable” proteins, including some well‐known oncoproteins such as KRas. Moreover, mutations of target genes limit the use of targeted drugs. Although some strategies such as RNA interference and DNA editing have been adopted to solve these problems, their clinical use remains challenging. Therefore, new strategies are urgently needed to meet the dilemmas encountered in the use of targeted drugs. As protein degradation is a rapid and well‐regulated biological process in cells, targeting protein degradation by inducing cellular protein degradation machinery, regardless of the status of the target, is an attractive idea for developing novel targeted drugs. This review summarizes recent advances in targeted protein degradation, with a focus on the current reagents and strategies used for inducing selective degradation of target proteins to provide clues for the development of novel anticancer drugs and cancer therapies.

Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts

The proteasome is an essential protein-degradation machinery in eukaryotic cells that controls protein turnover and thereby the biogenesis and function of cell organelles. Chloroplasts import thousands of nuclear-encoded precursor proteins from the cytosol, suggesting that the bulk of plastid proteins is transiently exposed to the cytosolic proteasome complex. Therefore, there is a cytosolic equilibrium between chloroplast precursor protein import and proteasomal degradation. We show here that a shift in this equilibrium, induced by mild genetic proteasome impairment, results in elevated precursor protein abundance in the cytosol and significantly increased accumulation of functional photosynthetic complexes in protein import-deficient chloroplasts. Importantly, a proteasome lid mutant shows improved photosynthetic performance, even in the absence of an import defect, signifying that functional precursors are continuously degraded. Hence, turnover of plastid precursors in the cytosol represents a mechanism to constrain thylakoid membrane assembly and photosynthetic electron transport.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

Regulation of the 20S Proteasome by a Novel Family of Inhibitory Proteins.

Aims: The protein degradation machinery plays a critical role in the maintenance of cellular homeostasis, preventing the accumulation of damaged or misfolded proteins and controlling the levels of regulatory proteins. The 20S proteasome degradation machinery, which predominates during oxidative stress, is able to cleave any protein with a partially unfolded region, however, uncontrolled degradation of the myriad of potential substrates is improbable. This study aimed to identify and characterize the regulatory mechanism that controls 20S proteasome-mediated degradation. Results: Using a bioinformatic screen based on known 20S proteasome regulators, we have discovered a novel family of 20S proteasome regulators, named catalytic core regulators (CCRs). These regulators share structural and sequence similarities, and coordinate the function of the 20S proteasome by affecting the degradation of substrates. The CCRs are involved in the oxidative stress response via Nrf2, organizing into a feed-forward loop regulatory circuit, with some members stabilizing Nrf2, others being induced by Nrf2, and all of them inhibiting the 20S proteasome. Innovation and Conclusion: These data uncover a new family of regulatory proteins that utilize a fine-tuned mechanism to carefully modulate the activity of the 20S proteasome, in particular under conditions of oxidative stress, ensuring its proper functioning by controlling the degradative flux.

Direct visualization of degradation microcompartments at the ER membrane

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.

Structural basis of antagonism of human APOBEC3F by HIV-1 Vif.

HIV-1 Vif promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of HIV-1 replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and its cellular cofactor CBFβ, at 3.9 Å resolution. The structure shows that Vif and CBFβ form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFβ beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

Inducible Phase Separation of GSK3α As a Mechanism for Asparaginase Resistance in Acute Leukemias

Asparaginase is an antileukemic enzyme that depletes the nonessential amino acid asparagine, but resistance is a common clinical problem whose biologic basis is poorly understood. We recently found that Wnt-induced inhibition of glycogen synthase kinase 3 (GSK3) profoundly sensitizes drug-resistant leukemias to asparaginase (Hinze et al, Cancer Cell, 2019;35:664). This effect is mediated by a β-catenin independent branch of Wnt signaling termed Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and proteasomal degradation (Acebron et al, Mol Cell, 2014;54:663). Thus, asparaginase-resistant leukemias rely on catabolic protein degradation as an alternative source of amino acids to survive asparaginase therapy. Asparaginase resistance is selectively mediated by GSK3α, because its genetic or pharmacologic inhibition fully phenocopied Wnt-induced sensitization to asparaginase (p < 0.0001), whereas selective inhibition of GSK3β had no effect. This is surprising because GSK3α and GSK3β are closely related paralogs thought to be redundant for many of their biologic functions. Thus, our objective was to define why asparaginase resistance is selectively dependent on GSK3α activity. To define the GSK3 domains responsible for asparaginase resistance, we leveraged the fact that selective depletion of GSK3α induces profound sensitization to asparaginase, and this effect is rescued by expression of a cDNA encoding GSK3α, but not GSK3β. We thus tested whether asparaginase resistance could be restored by expression of a series of GSK3 alleles in which the N-terminal, kinase, and C-terminal domains of GSK3α and GSK3β were swapped in various configurations. This revealed that asparaginase resistance is dependent on the N-terminal domain of GSK3α, whereas the kinase and the C-terminal domain were interchangeable. Fusing the N-terminus of GSK3α to the kinase and C-terminal domains of GSK3β fully restored asparaginase resistance in GSK3α depleted T-ALL (p < 0.0001) and AML cells (p < 0.0001). By contrast, fusing the N-terminus of GSK3β to the kinase and C-terminus of GSK3α had no discernible effect on response to asparaginase (p = n.s.). To investigate how the N-terminus of GSK3α regulates asparaginase response, we first applied structural prediction algorithms. This revealed that the N-terminal domain of GSK3α is a low-complexity (or prion-like) domain predicted to be intrinsically disordered, features associated with liquid-liquid phase separation. Phase separation is an increasingly recognized feature of cell biology that allows cells to concentrate components of important biochemical reactions in so-called membraneless organelles, thus promoting high-catalytic efficiency. Indeed, immunofluorescence confocal microscopy revealed that GSK3α, but not GSK3β, translocates into cytoplasmic bodies in response to asparagine depletion (p < 0.0001). The cytoplasmic GSK3a bodies were membraneless, as assessed by a proteinase K protection assay, and appeared to be distinct from known phase-separated compartments such as stress granules, P-bodies and aggresomes. However, cytoplasmic GSK3α bodies colocalized with the heat shock protein 70 (HSP70), K48-linked ubiquitin and the proteasome, suggesting that these bodies function in protein unfolding, ubiquitination and degradation. Indeed, genetic depletion of either GSK3α or HSP70 blocked formation of phase-separated GSK3α bodies (p < 0.0001) and induced asparaginase sensitivity (p < 0.0001). To explore the clinical relevance of GSK3α body formation in response to asparagine starvation, we tested a panel of matched asparaginase resistant vs. sensitive T-ALL, AML and B-ALL patient-derived xenografts (PDXs), and found that the ability of GSK3α to undergo phase separation significantly correlated with resistance to asparaginase in T-ALL, B-ALL and AML (Figure 1, p < 0.0001). Our data support a model in which inducible phase separation of GSK3α and heat shock proteins represents a previously unrecognized response to amino acid starvation that concentrates the cellular machinery for protein degradation, thus allowing efficient catalysis of this alternative source of amino acids in response to amino acid starvation. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Stevenson:Celgene: Research Funding. Neuberg:Celgene: Research Funding; Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership. Bourquin:Servier: Other: Travel support.

Quantitative PK/PD Prediction of the Efficacious and Safe Dose Ranges of the LMP7 Inhibitor M3258 for Phase I Application in Relapsed/Refractory Multiple Myeloma Patients

M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome; a crucial component of the cellular protein degradation machinery, which is highly expressed in malignant hematopoietic cells including multiple myeloma. M3258 was previously shown to deliver strong in vivo preclinical efficacy in multiple myeloma xenograft models, as well as a more benign non-clinical safety profile compared to approved pan-proteasome inhibitors, exemplified by a lack of effects on the central and peripheral nervous systems and cardiac and respiratory organs. Here we describe preclinical PK/PD and PK/efficacy modelling which led to a prediction of the PK profile, and the efficacious and safe dose ranges of M3258 in human which were used to guide the design of the phase I dose-escalation trial of M3258 in >3 line relapsed/refractory multiple myeloma (RRMM) patients. Mouse, rat, dog and monkey PK, plasma protein binding and intrinsic clearance data were used to estimate a half-life of approximately 6 hours for M3258 in human. The human total clearance and volume of distribution for M3258 were predicted to be 0.033 L/h/kg and 0.28 L/kg, respectively, whilst oral bioavailability was estimated to be above 80%. LMP7 proteolytic activity was assessed as a PD readout in human multiple myeloma tumor cells xenografted to mice as well as in dog peripheral blood mononuclear cells (PBMCs). A strong PK/PD relationship was observed for M3258 across both species. LMP7 inhibition by M3258 also correlated strongly with anti-tumor efficacy in multiple myeloma xenografts, with maximal efficacy observed at M3258 exposure delivering sustained inhibition of tumor LMP7 activity. Quantitative PK/PD/efficacy modeling predicted the biologically efficacious dose (BED) of M3258 upon oral application to be between 10 - 90 mg daily in human. By incorporating data from nonclinical safety studies, these data suggest an attractive human PK profile of M3258, enabling oral application, as well as an improved human therapeutic index compared to approved pan-proteasome inhibitors. M3258 is being investigated in a phase I, first-in-man, 2-part, open label clinical study designed to determine the safety, tolerability, PK, PD and early signs of efficacy of M3258 as a single agent (dose-escalation) and co-administered with dexamethasone (dose-expansion) in participants with RRMM whose disease has progressed following > 3 prior lines of therapy and for whom no effective standard therapy exists. Integration of these data will guide the selection of the BED for potential further clinical development of M3258. Disclosures Lignet: Merck Healthcare KGaA: Employment. Esdar:Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Becker:Merck Healthcare KGaA: Employment, Other: Holding shares with a value below 1000-USD. Drouin:EMD Serono Research and Development Institute: Employment. El Bawab:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Gimmi:Merck Healthcare KGaA: Employment. Haselmayer:Merck Healthcare KGaA: Employment. Jährling:Merck Healthcare KGaA: Employment. Sanderson:Merck Healthcare KGaA: Employment. Sloot:Merck Healthcare KGaA: Employment. Stinchi:Merck Healthcare KGaA: Employment. Victor:Merck Healthcare KGaA: Employment. Walter:Merck Healthcare KGaA: Employment. Rohdich:Merck Healthcare KGaA: Employment.

Cryo-EM structure of the ClpXP protein degradation machinery.

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXP’s complex mode of action and provides a structural framework for future pharmacological applications.

Quality Control in Huntington's Disease: a Therapeutic Target.

Huntington's disease (HD) is a fatal autosomal dominantly inherited brain disease caused by excessively expanded CAG repeats in gene which encodes huntingtin protein. These abnormally encoded huntingtin proteins and their truncated fragments result in disruption of cellular quality mechanism ultimately triggering neuronal death. Despite great efforts, a potential causative agent leading to genetic mutation in HTT, manifesting the neurons more prone to oxidative stress, cellular inflammation, energy depletion and apoptotic death, has not been established yet. Current scenario concentrates on symptomatic pathologies to improvise the disease progression and to better the survival. Most of the therapeutic developments have been converged to rescue the protein homeostasis. In HD, abnormal expansion of glutamine repeats in the protein huntingtin leads to toxic aggregation of huntingtin which in turn impairs the quality control mechanism of cells through damaging the machineries involved in removal of aggregated abnormal protein. Therapeutic approaches to improve the efficiency of aggregate clearance through quality control mechanisms involve protein folding machineries such as chaperones and protein degradation machineries such as proteasome and autophagy. Also, to reduce protein aggregation by enhancing proper folding, to degrade and eliminate the aggregates are suggested to negatively regulate the HD progression associated with the disruption of protein homeostasis. This review focuses on the collection of therapeutic strategies targeting enhancement of protein quality control activity to delay the HD pathogenesis.

Protein quality control machinery in intracellular protozoan parasites: hopes and challenges for therapeutic targeting.

Intracellular protozoan parasites have evolved an efficient protein quality control (PQC) network comprising protein folding and degradation machineries that protect the parasite's proteome from environmental perturbations and threats posed by host immune surveillance. Interestingly, the components of PQC machinery in parasites have acquired sequence insertions which may provide additional interaction interfaces and diversify the repertoire of their biological roles. However, the auxiliary functions of PQC machinery remain poorly explored in parasite. A comprehensive understanding of this critical machinery may help to identify robust biological targets for new drugs against acute or latent and drug-resistant infections. Here, we review the dynamic roles of PQC machinery in creating a safe haven for parasite survival in hostile environments, serving as a metabolic sensor to trigger transformation into phenotypically distinct stages, acting as a lynchpin for trafficking of parasite cargo across host membrane for immune evasion and serving as an evolutionary capacitor to buffer mutations and drug-induced proteotoxicity. Versatile roles of PQC machinery open avenues for exploration of new drug targets for anti-parasitic intervention and design of strategies for identification of potential biomarkers for point-of-care diagnosis.

Integration of Fungus-Specific CandA-C1 into a Trimeric CandA Complex Allowed Splitting of the Gene for the Conserved Receptor Exchange Factor of CullinA E3 Ubiquitin Ligases in Aspergilli.

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution.IMPORTANCEAspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.

Co-suppression of NbClpC1 and NbClpC2, chaperone subunits in the Clp protease complex, accelerates hypersensitive response and increases disease susceptibility in Nicotiana benthamiana

The Clp protease complex, which is the central protein degradation machinery in plastids, plays a role similar to proteasome. ClpC1 and ClpC2 comprise the chaperonic part of the Clp protease and share more than 90% nucleotide sequence similarity with each other. In a previous study, the co-suppression of Nicotiana benthamiana ClpC1 and ClpC2 (NbClpC1/C2) resulted in leaf chlorosis, growth retardation phenotype, disappearance of apical dominance and coarsely packed mesophyll cells. Quantitative real-time PCR analysis revealed that the co-suppression of NbClpC1/C2 upregulated the expression levels of defense-related genes including pathogenesis-related protein 1b (PR1b), glutathione-S-transferase, catalase, ascorbate peroxidase and systemic acquired resistance gene 8.2 (SAR8.2). NbClpC1/C2 co-suppressed leaves accelerated hypersensitive response by the infection with an incompatible pathogen, Pseudomonas syringae pv. tomato T1, but increased susceptibility via the infiltration of a compatible pathogen, P. syringae pv. tabaci. These findings indicate that disruption of the chaperones of ClpC1 and ClpC2 increases the growth of bacterial pathogens and therefore increases hypersensitive response and disease susceptibility.

Comparison of Drosophila melanogaster Embryo and Adult Proteome by SWATH-MS Reveals Differential Regulation of Protein Synthesis, Degradation Machinery, and Metabolism Modules.

An important area of modern biology consists of understanding the relationship between genotype and phenotype. However, to understand this relationship it is essential to investigate one of the principal links between them: the proteome. With the development of recent mass-spectrometry approaches, it is now possible to quantify entire proteomes and thus relate them to different phenotypes. Here, we present a comparison of the proteome of two extreme developmental states in the well-established model organism Drosophila melanogaster: adult and embryo. Protein modules such as ribosome, proteasome, tricarboxylic acid cycle, glycolysis, or oxidative phosphorylation were found differentially expressed between the two developmental stages. Analysis of post-translation modifications of the proteins identified in this study indicates that they generally follow the same trend as their corresponding protein. Comparison between changes in the proteome and the transcriptome highlighted patterns of post-transcriptional regulation for the subunits of protein complexes such as the ribosome and the proteasome, whereas protein from modules such as TCA cycle, glycolysis, and oxidative phosphorylation seem to be coregulated at the transcriptional level. Finally, the impact of the endosymbiont Wolbachia pipientis on the proteome of both developmental states was also investigated.

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.

Discovery of Protein Degradation Machinery at the Desmosome Reveals Novel Triggers of the Desmosomal Disease, Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic‐based cardiac disease which is characterized by right/bi‐ventricular dysfunction, fibro‐fatty replacement of the myocardium and ventricular arrhythmias, leading to sudden death in young people and athletes. ARVC is termed a “disease of the desmosome” (mechanical cell‐cell junction) as 40% of ARVC patients harbor mutations and/or loss of desmosomal components. However, limited information exists on how cardiac desmosomal protein loss/degradation triggers ARVC. Through a yeast‐two‐hybrid screen in an adult human heart cDNA library, we uncovered a new interaction between the desmosomal protein, desmoplakin (DSP, N terminus) and a protein degradation machinery component, CSN6 (COP9 signalosome subunit 6). Traditionally, CSN proteins are thought to prevent proteins from being degraded; however, a role for CSN6 in the heart is undefined. We show that CSN6 localizes to desmosomal junctions in cardiac muscle and structurally complex with desmoplakin, suggesting that the novel hypothesis that CSN6 may protect the cardiac desmosome from degradation and loss/destablization this pathway may lead to ARVC. Using two novel cardiac‐specific CSN6 knockout (CSN6‐KO) mice, we further show that CSN6 loss accelerates desmosome destruction and triggers ARVC, as CSN6‐KO hearts exhibit selective loss of desmosomal proteins and recapitulate the key cardiomyopathy features and sudden death associated with ARVC, similar to classic mouse model of ARVC (DSP‐cKO). CSN6‐KO and DSP‐cKO hearts also harbor underlying hyper‐accumulation of protein degradation machinery specifically at cell‐cell junctions. Restoration of CSN function by the protein degradation inhibitor (MLN4924) could rescue the loss of desmosomal protein expression associated with CSN6 loss in vitro, further highlighting that the cardiac desmosome is regulated by protein degradation. CSN6 pathways are also relevant to human ARVC as a N‐terminus desmoplakin mutation (R315C) identified in a human ARVC patient was sufficient to abrogate DSP‐CSN6 binding and result in loss of CSN6 localization at the cell‐cell junction in cardiac biopsies. DSP R315C knock‐in mice also exhibited cardiac CSN6 reduction and early electrical defects found in ARVC. We altogether identify CSN6 as a novel “brake” to prevent desmosomal protein degradation, and reveal that disruption of this complex is an important “accelerator” of sudden death and ARVC.Support or Funding InformationAmerican Heart Association Postdoctoral Fellowship 17POST33670205This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.