Structural basis of TFIIIC-dependent RNA polymerase III transcription initiation.

RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.

The discovery of zinc fingers and their applications in gene regulation and genome manipulation.

An account is given of the discovery of the classical Cys(2)His(2) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of structural studies on its structure and its interaction with DNA. The finger is a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design is ideally suited for engineering proteins to target specific genes. In the first example of their application in 1994, a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In addition, a reporter gene was activated by targeting an inserted zinc finger promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that by combining zinc fingers with other effector domains, e.g., from nucleases or integrases, to form chimeric proteins, genomes could be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of therapeutic importance.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

Proteomic analysis of aged and OPTN E50K retina in the development of normal tension glaucoma.

Progressive degeneration of retinal ganglion cells (RGCs) is a major characteristic of glaucoma, whose underlying mechanisms are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal tension glaucoma (NTG), directly affecting RGCs without high intraocular pressure and causing severe glaucomatous symptoms in clinical settings. A systematic analysis of the NTG mouse model is crucial for better understanding of the underlying pathological mechanisms for glaucoma. To elucidate proteomic and biochemical pathway alterations during NTG development, we established an OPTN E50K mutant mouse model through CRISPR/Cas9. Retinal proteins from resulting mice exhibiting glaucomatous phenotypes were subject to tandem mass tag-labeled quantitative proteomics and then analyzed through bioinformatics methods to characterize the molecular and functional signatures of NTG. We identified 6364 quantitative proteins in our proteomic analysis. Bioinformatics analysis revealed that OPTN E50K mice experienced protein synthesis dysregulation, age-dependent energy defects and autophagy-lysosome pathway dysfunction. Certain biological features, including amyloid deposition, RNA splicing, microglia activation and reduction of crystallin production, were similar to Alzheimer's disease. Our study is the first to describe proteomic and biochemical pathway alterations in NTG pathogenesis during disease advancement. Several proteomic signatures overlapped with retinal changes found in the ad mice model, suggesting the presence of common mechanisms between age-related degenerative disorders, as well as prospective new targets for diagnostic and therapeutic strategies.

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy.

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway.

Knockdown of optineurin controls C2C12 myoblast differentiation via regulating myogenin and MyoD expressions.

Mutations in optineurin (OPTN) have been identified in a small proportion of sporadic and familial amyotrophic lateral sclerosis (ALS) cases. Recent evidences suggest that OPTN would be involved in not only the pathophysiological mechanisms of motor neuron death of ALS but also myofiber degeneration of sporadic inclusion body myositis. However, the detailed role of OPTN in muscle remains unclear. Initially, we showed that OPTN expression levels were significantly increased in the denervated muscles of mice, suggesting that OPTN may be involved in muscle homeostasis. To reveal the molecular role of OPTN in muscle atrophy, we used cultured C2C12 myotubes treated with tumor necrosis factor-like inducer of apoptosis (TWEAK) as an in vitro model of muscle atrophy. Our data showed that OPTN had no effect on the process of muscle atrophy in this model. On the other hand, we found that myogenic differentiation was affected by OPTN. Immunoblotting analysis showed that OPTN protein levels gradually decreased during C2C12 differentiation. Furthermore, OPTN knockdown inhibited C2C12 differentiation, accompanied by reduction of mRNA and protein expression levels of myogenin and MyoD. These findings suggested that OPTN may have a novel function in muscle homeostasis and play a role in the pathogenesis of neuromuscular diseases.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

OPTN recruitment to a Golgi-proximal compartment regulates immune signalling and cytokine secretion.

Optineurin (OPTN) is a multifunctional protein involved in autophagy and secretion, as well as nuclear factor κB (NF-κB) and IRF3 signalling, and OPTN mutations are associated with several human diseases. Here, we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-κB and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations in OPTN linked to primary open-angle glaucoma (POAG) cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. By using proximity labelling proteomics, we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-κB and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment, leading to altered pro-inflammatory cytokine secretion.

Zebrafish optineurin: genomic organization and transcription regulation.

Optineurin (OPTN) is involved in a variety of mechanisms, such as autophagy, vesicle trafficking, and nuclear factor kappa-B (NF-κB) signaling. Mutations in the OPTN gene have been associated with different pathologies, including glaucoma, amyotrophic lateral sclerosis, and Paget's disease of bone. Since the relationship between fish and mammalian OPTN is not well understood, the objective of the present work was to characterize the zebrafish optn gene and protein structure and to investigate its transcriptional regulation. Through a comparative in silico analysis, we observed that zebrafish optn presents genomic features similar to those of its human counterpart, including its neighboring genes and structure. A comparison of OPTN protein from different species revealed a high degree of conservation in its functional domains and three-dimensional structure. Furthermore, our in vitro transient-reporter analysis identified a functional promoter in the upstream region of the zebrafish optn gene, along with a region important for its transcription regulation. Site-directed mutagenesis revealed that the NF-κB motif is responsible for the activation of this region. In conclusion, with this study, we characterize zebrafish optn and our results indicate that zebrafish can be considered as an alternative model to study OPTN's biological role in bone-related diseases.

Molecular Dynamics Simulations of Reduced and Oxidized TFIIIA Zinc Fingers Free and Interacting with 5S RNA.

Interactions of zinc finger (ZF) proteins with nucleic acids and proteins play an important role in DNA transcription and repair, biochemical recognition, and protein regulation. The release of Zn2+ through oxidation of cysteine thiolates is associated with disruption of gene expression and DNA repair, preventing tumor growth. Multi-microsecond molecular dynamics (MD) simulations were carried out to examine the effect of Cys oxidation on the ZF456 fragment of transcription factor III A (TFIIIA) and its complex with 5S RNA. In the absence of 5S RNA, the reduced ZF456 peptide undergoes conformational changes in the secondary structure due to the reorientation of the intact ZF domains. Upon oxidation, the individual ZF domains unfold to various degrees, yielding a globular ZF456 peptide with ZF4 and ZF6, responsible for base-specific hydrogen bonds with 5S RNA, losing their ßßα-folds. ZF5, on the other hand, participates in nonspecific interactions through its α-helix that conditionally unravels early in the simulation. In the presence of RNA, oxidation of the ZF456 peptide disrupts the key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. However, interactions with ZF4 are dependent on the protonation state of His119.

OPTN variants in ALS cases: a case report of a novel mutation and literature review.

INTRODUCTION: Optineurin (OPTN)-associated mutations have been implicated in the development of type 12 amyotrophic lateral sclerosis (ALS12). We reported a case of ALS with a new OPTN variant (p.D527fs) and reviewed relevant literature to better understand the phenotypes and pathophysiological mechanisms of ALS12. METHODS: We report a case of a 55-year-old female patient with a new heterozygous variant of the OPTN gene. A literature search of ALS cases associated with the OPTN gene mutations was performed in PubMed with the search criteria as [("amyotrophic lateral sclerosis") OR ("motor neuron disease")] AND ("OPTN"). RESULTS: The case of ALS with a new OPTN variant (p.D527fs) in our report manifested with bulbar involvement in onset and a rapidly progressive course. A literature review of 37 ALS patients with OPTN mutations included 20 males and 16 females with another patient whose gender was not described. The mean onset age of 37 ALS12 patients was 48 with the youngest 23 and the oldest 83 years old. Differences in onset age between male and female patients were not significant. Mean time from initiation to death was 61.8 ± 12.0 months. Patients present with either limb onset (73.5% cases) or bulbar onset (23.5% cases). CONCLUSION: Through the literature review, we summarized the clinical characteristics of ALS12. The phenotypes of the reported patients elucidate the genetic profiles and clinical phenotypes of ALS12. Clinicians should pay close attention to the role of receptor-interacting kinase 1 (RIPK1)-dependent necroptosis in the pathophysiologic development of ALS12, since necroptosis inhibitors are expected as potential therapeutic agents for treating ALS12.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection.

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

Yeast genetic interaction screen of human genes associated with amyotrophic lateral sclerosis: identification of MAP2K5 kinase as a potential drug target.

To understand disease mechanisms, a large-scale analysis of human-yeast genetic interactions was performed. Of 1305 human disease genes assayed, 20 genes exhibited strong toxicity in yeast. Human-yeast genetic interactions were identified by en masse transformation of the human disease genes into a pool of 4653 homozygous diploid yeast deletion mutants with unique barcode sequences, followed by multiplexed barcode sequencing to identify yeast toxicity modifiers. Subsequent network analyses focusing on amyotrophic lateral sclerosis (ALS)-associated genes, such as optineurin (OPTN) and angiogenin (ANG), showed that the human orthologs of the yeast toxicity modifiers of these ALS genes are enriched for several biological processes, such as cell death, lipid metabolism, and molecular transport. When yeast genetic interaction partners held in common between human OPTN and ANG were validated in mammalian cells and zebrafish, MAP2K5 kinase emerged as a potential drug target for ALS therapy. The toxicity modifiers identified in this study may deepen our understanding of the pathogenic mechanisms of ALS and other devastating diseases.

Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression.

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.

Optineurin promotes autophagosome formation by recruiting the autophagy-related Atg12-5-16L1 complex to phagophores containing the Wipi2 protein.

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

A critical role of Hrd1 in the regulation of optineurin degradation and aggresome formation.

Mutations in optineurin (OPTN) are associated with several human disorders including amyotrophic lateral sclerosis (ALS) and primary open-angle glaucoma (POAG). OPTN is known to be a multifunctional autophagy receptor that plays important roles in NF-κB signaling, vesicle trafficking, maintenance of the Golgi apparatus and autophagy. Given that a loss of neurons and an abnormal aggregation of disease proteins are two key features of neurodegenerative diseases, protein quality control systems are considered to be tightly associated with neurodegeneration. In this study, we investigated the involvement of the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway, two major intracellular protein quality control systems, in the regulation of wild-type (WT) OPTN, ALS-linked mutant E478G OPTN and POAG-linked mutant E50K OPTN. Our data revealed that the UPS, not the autophagy-lysosome pathway, is the major system for degradation and aggregation of OPTN. Moreover, we found that Hrd1, an E3 ubiquitin ligase, could play an important role in the protein quality control of OPTN. Our results demonstrated that overexpression of Hrd1 increased the proteasomal degradation and microtubule-dependent aggresome formation of OPTN in the microtubular organizing center, whereas knockdown of Hrd1 stabilized OPTN and inhibited aggresome formation of OPTN.