Defective Protein Degradation Research Articles

Mucopolysaccharidosis type II zebrafish model exhibits early impaired proteasomal-mediated degradation of the axon guidance receptor Dcc.

Most of the patients affected by neuronopathic forms of Mucopolysaccharidosis type II (MPS II), a rare lysosomal storage disorder caused by defects in iduronate-2-sulfatase (IDS) activity, exhibit early neurological defects associated with white matter lesions and progressive behavioural abnormalities. While neuronal degeneration has been largely described in experimental models and human patients, more subtle neuronal pathogenic defects remain still underexplored. In this work, we discovered that the axon guidance receptor Deleted in Colorectal Cancer (Dcc) is significantly dysregulated in the brain of ids mutant zebrafish since embryonic stages. In addition, thanks to the establishment of neuronal-enriched primary cell cultures, we identified defective proteasomal degradation as one of the main pathways underlying Dcc upregulation in ids mutant conditions. Furthermore, ids mutant fish-derived primary neurons displayed higher levels of polyubiquitinated proteins and P62, suggesting a wider defect in protein degradation. Finally, we show that ids mutant larvae display an atypical response to anxiety-inducing stimuli, hence mimicking one of the characteristic features of MPS II patients. Our study provides an additional relevant frame to MPS II pathogenesis, supporting the concept that multiple developmental defects concur with early childhood behavioural abnormalities.

Proteomics approach reveals urinary markers for early pregnancy diagnosis in buffaloes

This study aimed to compare urine proteomics from non- and pregnant buffaloes in order to identify potential biomarkers of early pregnancy. Forty-four females underwent hormonal ovulation synchronization and were randomly divided into two experimental groups: inseminated (n = 30) and non-inseminated (n = 14). The pregnant females were further divided into two groups: pregnant at Day 12 (P12; n = 8) and at Day 18 (P18; n = 8) post-ovulation. The non-pregnant group was also subdivided into two groups: non-pregnant at Day 12 (NP12; n = 7) and at Day 18 (NP18; n = 7). Urine was collected from all females on Days 12 or 18. The samples were processed for proteomics. A total of 798 proteins were reported in the urine considering all groups. The differential proteins play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that some proteins from our study can be considered biomarkers for early pregnancy diagnosis, since they were increased in pregnant buffaloes. SignificanceMacromolecules have been studied for early pregnancy diagnosis, aiming to increase reproductive efficiency in cattle and buffaloes. Direct methods such as rectal palpation and ultrasonography have been considered late. Thus, this study aimed to compare urine proteomics from non- and pregnant buffaloes to identify potential biomarkers of early pregnancy. The differential proteins found in our study play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that these proteins can be considered possible biomarkers for early pregnancy diagnosis since they were increased in the pregnant buffaloes.

Neuropathological outcomes of traumatic brain injury and alcohol use in males and females: studies using preclinical rodent and clinical human specimens.

Traumatic brain injury (TBI) and alcohol misuse are inextricably linked and can increase the risk for developing neurodegenerative diseases, particularly in military veterans and contact sport athletes. Proteinopathy (defects in protein degradation) is considered an underlying factor in neurodegenerative diseases. However, whether it contributes to TBI/alcohol-mediated neurodegeneration is unexplored. Our recent studies have identified ISGylation, a conjugated form of ISG15 (Interferon-Stimulated Gene 15) and inducer of proteinopathy, as a potential mechanistic link underlying TBI-mediated neurodegeneration and proteinopathy in veterans. In the current study, a rat model of combined TBI and alcohol use was utilized to investigate the same relationship. Here, we report sustained induction of Interferon β (IFNβ), changes in TAR DNA Binding 43 (TDP-43) ISGylation levels, TDP-43 proteinopathy (C-terminal fragmentation (CTF)), and neurodegeneration in the ventral horns of the lumbar spinal cords (LSCs) and/or motor cortices (MCs) of female rats post-TBI in a time-dependent manner. In males, these findings mostly remained non-significant, though moderate alcohol use appears to decrease neurodegeneration in males (but not females) post-TBI. We, however, do not claim that moderate alcohol consumption is beneficial for preventing TBI-mediated neurodegeneration. We have previously demonstrated that ISGylation is increased in the LSCs of veterans with TBI/ALS (Amyotrophic Lateral Sclerosis). Here, we show increased ISGylation of TDP-43 in the LSCs of TBI/ALS-afflicted female veterans compared to male veterans. Knowing that ISGylation induces proteinopathy, we suggest targeting ISGylation may prevent proteinopathy-mediated neurodegeneration post-TBI, particularly in women, however, causal studies are required to confirm this claim.

Defective protein degradation in genetic disorders

Understanding the molecular mechanisms that underlie different human pathologies is necessary to develop novel therapeutic strategies. An emerging mechanism of pathogenesis in many genetic disorders is the dysregulation of protein degradation, which leads to the accumulation of proteins that are responsible for the disease phenotype. Among the different cellular pathways that regulate active proteolysis, the Cullin RING E3 ligases represent an important group of sophisticated enzymatic complexes that mediate substrate ubiquitination through the interaction with specific adaptors. However, pathogenic variants in these adaptors affect the physiological ubiquitination of their substrates. This review discusses our current understanding of this emerging field.

The roles, controversies, and combination therapies of autophagy in lung cancer.

Lung cancer is one of the leading causes of death among men and women worldwide. The disease initially has a silent phenotype, which leads to the progression of the disease and ultimately the lack of proper response to routine treatments. Autophagy, known as an intracellular "recycle bin" for the degradation of defective proteins and molecules, is one of the mechanisms that has been considered in the context of cancer in recent years. This study aims to provide a comprehensive review of published articles on autophagy in the context of lung cancer to have a complete view of the role of autophagy in lung cancer and its possible treatments. PubMed, Scopus, and Google Scholar were searched until June 15 to find related articles. No specific search filters or restrictions were applied. The results were entered into reference management software for aggregation and management. The full text of all articles was screened and studied. In conclusion, studies on the exact function of autophagy in lung cancer are contradictory, but what can be concluded from a review of literature on lung cancer is that targeting autophagy combined with traditional routine therapies such as chemotherapy, especially in advanced stages of lung cancer, can be an effective anticancer approach.

Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes.

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

ALS/FTD-causing mutation in cyclin F causes the dysregulation of SFPQ.

Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.

Treatment Options for Coronavirus Disease 2019 in Patients With Reduced or Absent Kidney Function.

Coronavirus disease 2019, the disease caused by the severe acute respiratory syndrome coronavirus 2 virus, was first identified in the Hubei Province of China in late 2019. Currently, the only role for therapy is treatment of the disease, as opposed to postexposure prophylaxis, however multiple clinical trials are currently ongoing for both treatment and prophylaxis. Treating coronavirus disease 2019 relies on two components; the first is inhibition of the viral entrance and replication within the body and the second is inhibition of an exacerbated immune response which can be seen in patients with severe disease. Many drugs have shown in vitro antiviral activity; however, clinical trials have not been as promising. This review summarizes the current data for the most commonly used drugs for coronavirus disease 2019 and will cover the unique factors that may affect the dosing of these medications in patients with CKD. While clinical trials are ongoing, most are in patients with normal kidney function. During a pandemic, when patients with CKD are at higher risk of both infection and death, it is imperative to include patients these patients in the clinical trials.

Proteomic Signatures Reveal Differences in Stress Response, Antioxidant Defense and Proteasomal Activity in Fertile Men with High Seminal ROS Levels.

Elevated levels of reactive oxygen species (ROS) are a major cause of male infertility. However, some men with high seminal ROS levels are still fertile. The main objective of this study was to understand the molecular mechanism(s) responsible for the preservation of fertility in those men. Semen samples from fertile men were divided into two groups: control (n = 10, ROS < 102.2 RLU/s/106 sperm) and ROS+ (n = 10, ROS > 102.2 RLU/s/106 sperm). Proteomic analysis of seminal plasma and spermatozoa was used to identify the differentially expressed proteins (DEPs) between the experimental groups, from which some proteins were validated by Western blot (WB). A total of 44 and 371 DEPs were identified between the study groups in the seminal plasma and spermatozoa, respectively. The identified DEPs were primarily involved in oxidoreductase, endopeptidase inhibitor, and antioxidant activities. We validated by WB the underexpression of NADH:ubiquinone oxidoreductase core subunit S1 (p = 0.01), as well as the overexpression of superoxide dismutase 1 (p = 0.03) and peroxiredoxin 4 (p = 0.04) in spermatozoa of ROS+ group. Our data suggest that fertile men with high ROS levels possess an effective antioxidant defense system that protects sperm proteins, as well as an active proteasomal system for degradation of defective proteins.

Tissue Localization of Glycosphingolipid Accumulation in a Gaucher Disease Mouse Brain by LC-ESI-MS/MS and High-Resolution MALDI Imaging Mass Spectrometry

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

Abstract 2770: DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism

Abstract Physiological cellular activity requires a precise control of the proteome. However, cancer cells have a distorted protein profile. It is likely that intrinsic defects in protein homeostasis participate in human tumorigenesis, such as alterations in de novo protein synthesis, or defects in protein degradation. In this last case, the ubiquitin (Ub) proteasome system (UPS) targets a variety of proteins, including functional proteins that are no longer needed. Without appropriate protein homeostasis maintained by the UPS, healthy cells can undergo malignant transformation, and this observation has been therapeutically exploited by the development of proteasome inhibitors as anticancer agents. Many of the cytoplasm UPS-degraded proteins are retrotranslocated from the endoplasmic reticulum (ER). In this regard, ER possesses a quality control mechanism, termed the ER-associated degradation mechanism (ERAD), that is induced in response to ER stress by a transcriptional program, known as the unfolded protein response (UPR), which leads to the accelerated degradation of unfolded proteins. Within the ERAD pathway, the Derlin proteins play a critical role. It has been proposed that Derlins form an export channel in the membrane of the ER through which the ERAD substrates pass to reach the proteasome. A previous study in our lab has shown that DERL3 silencing by DNA methylation deregulate SLC2A1 (GLUT1) degradation, promoting the Warburg effect. SLC2A1 is a key glucose receptor up-regulated in most solid tumors, allowing cancer cells to get the glucose they need for tumor progression. In this study we use an embryonic rhabdomyosarcoma cell line as a model to find out the role of DERL3 in these tumors, as the percentage of promoter hypermethylation is these tumors is approximately 60%. Rhabdomyosarcoma (RMS) is one of the leading causes of cancer related deaths among children, being the histological variant of embryonic rhabdomyosarcoma the most common soft tissue sarcoma of childhood and adolescence, with 350 cases per year in USA. The restoration of DERL3 expression in vitro promotes GLUT1 degradation and GLUT4 up-regulation, becoming cells sensitive to insulin. Moreover, it has been shown that DERL3 confers sensitivity against an ERAD related drug, Eeyarestatin I, opening a new approach for RMS treatment. All together, these initial findings highlight the role of DERL3 in RMS metabolism. Citation Format: Pere Llinas, Paula Lopez, Manel Esteller. DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2770.

FBXO10 deficiency and BTK activation upregulate BCL2 expression in mantle cell lymphoma.

Targeting Bruton tyrosine kinase (BTK) by ibrutinib is an effective treatment for patients with relapsed/refractory mantle cell lymphoma (MCL). However, both primary and acquired resistance to ibrutinib have developed in a significant number of these patients. A combinatory strategy targeting multiple oncogenic pathways is critical to enhance the efficacy of ibrutinib. Here, we focus on the BCL2 anti-apoptotic pathway. In a tissue microarray of 62 MCL samples, BCL2 expression positively correlated with BTK expression. Increased levels of BCL2 were shown to be due to a defect in protein degradation because of no or little expression of the E3 ubiquitin ligase FBXO10, as well as transcriptional upregulation through BTK-mediated canonical nuclear factor-κB activation. RNA-seq analysis confirmed that a set of anti-apoptotic genes (for example, BCL2, BCL-XL and DAD1) was downregulated by BTK short hairpin RNA. The downregulated genes also included those that are critical for B-cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Targeting BCL2 by the specific inhibitor ABT-199 synergized with ibrutinib in inhibiting growth of both ibrutinib-sensitive and -resistant cancer cells in vitro and in vivo. These results suggest co-targeting of BTK and BCL2 as a new therapeutic strategy in MCL, especially for patients with primary resistance to ibrutinib.

Metallo-pathways to Alzheimer's disease: lessons from genetic disorders of copper trafficking.

Copper is an essential metal ion that provides catalytic function to numerous enzymes and also regulates neurotransmission and intracellular signaling. Conversely, a deficiency or excess of copper can cause chronic disease in humans. Menkes and Wilson disease are two rare heritable disorders of copper transport that are characterized by copper deficiency and copper overload, respectively. Changes to copper status are also a common feature of several neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS). In the case of AD, which is characterized by brain copper depletion, changes in the distribution of copper has been linked with various aspects of the disease process; protein aggregation, defective protein degradation, oxidative stress, inflammation and mitochondrial dysfunction. Although AD is a multifactorial disease that is likely caused by a breakdown in multiple cellular pathways, copper and other metal ions such as iron and zinc play a central role in many of these cellular processes. Pioneering work by researchers who have studied relatively rare copper transport diseases has shed light on potential metal ion related disease mechanisms in other forms of neurodegeneration such as AD.

Yeast Importin-α (Srp1) Performs Distinct Roles in the Import of Nuclear Proteins and in Targeting Proteasomes to the Nucleus

Srp1 (importin-α) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-α mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.

Abstract 1343: Non-apoptotic death induced in glioma cells by the indole-based chalcone, MOMIPP, involves disruption of endolysosomal trafficking and ER stress

Abstract MOMIPP, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one, is a novel chalcone-related compound that induces non-apoptotic cell death in various cancer cell lines, including temozolomide-resistant glioblastoma (GBM) cells. The unique mechanism of cell death induced by MOMIPP has been termed “methuosis”. Morphological hallmarks of methuosis include early accumulation of large cytoplasmic vacuoles derived from macropinosomes, followed by detachment of cells from the substratum and necrosis-like rupture of the plasma membrane. Nuclear changes typical of apoptosis are absent. The targets of MOMIPP have not yet been identified and the mechanistic link between MOMIPP-induced vacuolization and cell death is poorly understood. Previous studies have shown that the non-clathrin coated macropinosome-derived vacuoles induced by MOMIPP fail to merge with lysosomes. In the present study we test the hypothesis that the cytotoxic activity of MOMIPP may entail perturbations of additional trafficking pathways that interface with lysosomes. We report that the post-translation proteolytic processing of pro-cathepsin D, which occurs in conjunction with trafficking of the pro-enzyme from late endosomes to lysosomes, is impaired in MOMIPP-treated GBM cells. In parallel, ligand-dependent turnover of the epidermal growth factor receptor is also attenuated. These findings are consistent with a global block in protein trafficking between late endosomes and lysosomes. Examining the lysosome-dependent process of macroautophagy, we found that MOMIPP caused an accumulation of the autophagosome marker, LC3-II, and decline in degradation of the autophagosome cargo, p62, consistent with a block in autophagosome-lysosome flux. Potentially related to the defects in protein degradation, we detected a robust activation of ER stress, as measured by increased expression of CHOP and phosphorylated-PERK in MOMIPP treated cells. In conjunction with the altered trafficking events, we observed swelling of acridine orange-positive acidic vesicles, in addition to the previously reported vacuolization of non-acidic macropinosomes. More precise identification of these compartments is in progress. Taken together, these results suggest that the induction of non-apoptotic cell death by MOMIPP may be related, at least in part, to dysfunctional trafficking at the nexus where macropinosomes, late endosomes and autophagosomes converge on lysosomes. Supported by NIH grant R01 CA115495 and the Harold and Helen McMaster Endowment Citation Format: Nneka E. Mbah, Andrew D. Trunk, Bryan A. Dewitt, Jean H. Overmeyer, William A. Maltese. Non-apoptotic death induced in glioma cells by the indole-based chalcone, MOMIPP, involves disruption of endolysosomal trafficking and ER stress. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2014-1343

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila

The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila

The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

P62 expression and autophagy in αB-crystallin R120G mutant knock-in mouse model of hereditary cataract

The formation of cataracts is associated with the accumulation of protein aggregates in the ocular lens, suggesting that defective protein degradation plays a role in cataract pathogenesis. Accumulation of the p62 protein has recently been identified as a marker for impaired autophagy in a variety of tissues; however, little information exists on its expression in the ocular lens and in cataracts. In the present study we examined the expression of p62 in the mouse lens and compared its expression in wild-type lenses with that in lenses from knock-in mice with an arginine to glycine mutation in αB-crystallin (αB-R120G) that is known to cause human hereditary cataract. Immunohistochemical, immunoblotting, and transmission electron microscopic analyses of wild type and αB-R120G mutant mice were performed. To assess the effect of increased protein aggregation on autophagy, immunohistochemical staining was performed with an anti-p62 antibody, revealing the presence of p62-positive punctate staining in a band of denucleated cortical fiber cells. The number and size of p62 puncta were significantly greater in αB-R120G homozygous mutant lenses than in wild type and heterozygous mutant lenses. p62 staining was also abundant in lens epithelial cells and was concentrated around the nuclear membrane. Double-membraned structures similar to autophagosomes containing cellular cytoplasmic content were detected in lens epithelial cells by transmission electron microscopy. The autophagosomes in lens epithelial cells from αB-R120G homozygous mutant mice were larger than those in wild type mice. Double-membraned structures that are probably autophagosomes were also detected in cortical fiber cells and were more abundant in the αB-R120G homozygous mutant lens than the wild type lens. This study demonstrates p62 distribution as speckles in the lens fiber cells, altered levels of p62 expression, and the presence of autophagosomes in the ocular lens of αB-R120G mutant mice. We propose that autophagy is inhibited in the αB-R120G mutant lenses because of a defect in protein degradation after autophagosome formation. Further work is necessary to determine the relationship between αB-crystallin function, autophagy, and cataract formation.

Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.