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.

Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy.

Optineurin is a multifunctional adaptor protein intimately involved in various vesicular trafficking pathways. Through interactions with an array of proteins, such as myosin VI, huntingtin, Rab8, and Tank-binding kinase 1, as well as via its oligomerisation, optineurin has the ability to act as an adaptor, scaffold, or signal regulator to coordinate many cellular processes associated with the trafficking of membrane-delivered cargo. Due to its diverse interactions and its distinct functions, optineurin is an essential component in a number of homeostatic pathways, such as protein trafficking and organelle maintenance. Through the binding of polyubiquitinated cargoes via its ubiquitin-binding domain, optineurin also serves as a selective autophagic receptor for the removal of a wide range of substrates. Alternatively, it can act in an ubiquitin-independent manner to mediate the clearance of protein aggregates. Regarding its disease associations, mutations in the optineurin gene are associated with glaucoma and have more recently been found to correlate with Paget's disease of bone and amyotrophic lateral sclerosis (ALS). Indeed, ALS-associated mutations in optineurin result in defects in neuronal vesicular localisation, autophagosome-lysosome fusion, and secretory pathway function. More recent molecular and functional analysis has shown that it also plays a role in mitophagy, thus linking it to a number of other neurodegenerative conditions, such as Parkinson's. Here, we review the role of optineurin in intracellular membrane trafficking, with a focus on autophagy, and describe how upstream signalling cascades are critical to its regulation. Current data and contradicting reports would suggest that optineurin is an important and selective autophagy receptor under specific conditions, whereby interplay, synergy, and functional redundancy with other receptors occurs. We will also discuss how dysfunction in optineurin-mediated pathways may lead to perturbation of critical cellular processes, which can drive the pathologies of number of diseases. Therefore, further understanding of optineurin function, its target specificity, and its mechanism of action will be critical in fully delineating its role in human disease.

Detection of mutations in MYOC, OPTN, NTF4, WDR36 and CYP1B1 in Chinese juvenile onset open-angle glaucoma using exome sequencing.

Juvenile onset open-angle glaucoma (JOAG) affects patients before 40 years of age, causing high intraocular pressure and severe optic nerve damage. To expand the mutation spectrum of the causative genes in JOAG, with a view to identify novel disease-causing mutations, we investigated MYOC, OPTN, NTF4, WDR36 and CYP1B1 in a cohort of 67 unrelated Chinese JOAG patients. Whole exome sequencing was used to identify possible pathogenic mutations, which were further excluded in normal controls. After sequencing and the use of a database pipeline, as well as predictive assessment filtering, we identified a total of six mutations in three genes, MYOC, OPTN and CYP1B1. Among them, 2 heterozygous mutations in MYOC (c. 1109C > T, p. (P370L); c. 1150G > C, p. (D384H)), 2 heterozygous mutations in OPTN (c. 985A > G, p.(R329G); c. 1481T > G, p. (L494W)) and 2 homozygous mutations in CYP1B1 (c. 1412T > G, p.(I471S); c. 1169G > A, p.(R390H)) were identified as potentially causative mutations. No mutation was detected in NTF4 or WDR36. Our results enrich the mutation spectra and frequencies of MYOC, OPTN and CYP1B1 in JOAG among the Chinese population. Further studies are needed to address the pathogenicity of each of the mutations detected in this study.

Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideß7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideß7 is a promising candidate for in vivo use and therapeutic applications.

Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis.

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-κB suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-κB suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-α-mediated NF-κB activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-α-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-κB are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-κB activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS.

Loss of optineurin in vivo results in elevated cell death and alters axonal trafficking dynamics.

Mutations in Optineurin have been associated with ALS, glaucoma, and Paget's disease of bone in humans, but little is known about how these mutations contribute to disease. Most of the cellular consequences of Optineurin loss have come from in vitro studies, and it remains unclear whether these same defects would be seen in vivo. To answer this question, we assessed the cellular consequences of Optineurin loss in zebrafish embryos to determine if they showed the same defects as have been described in the in vitro studies. We found that loss of Optineurin resulted in increased cell death, as well as subtle cell morphology, cell migration and vesicle trafficking defects. However, unlike experiments on cells in culture, we found no indication that the Golgi apparatus was disrupted or that NF-κB target genes were upregulated. Therefore, we conclude that in vivo loss of Optineurin shows some, but not all, of the defects seen in in vitro work.

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase.

Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)2-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes.

[C2H2 zinc-finger recognition of biomolecules].

C2H2 zinc-finger motif presents in 3% of proteins that are encoded in the human genome, and has the abilities to recognize DNA, RNA and protein. With nearly 3 decades of efforts, the mechanisms of zinc-finger mediated biomolecule recognitions have been studied to various extents. Zinc-finger binds into the major groove of DNA double helix, establishes an one-to-one recognition format between DNA bases and certain amino acids in a zinc-finger, and achieves specificity based on DNA sequences. While RNA molecules show a large variety in their structures, zinc-finger recognizes RNA through the collected information of specially displayed bases and special backbone folding. Initial studies have been performed on zinc-finger mediated protein-protein interactions. Existing data indicate multiple recognition modes. The studies on molecular mechanism have supported the development of engineered zinc-fingers, which have been introduced into applications. For its wide existence, large functional diversity and potential in translational applications, zinc-finger deserves a systematic study in every aspect.

Structural basis for phosphorylation-triggered autophagic clearance of Salmonella.

Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys5¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.

Enhanced optineurin E50K-TBK1 interaction evokes protein insolubility and initiates familial primary open-angle glaucoma.

Glaucoma is the leading cause for blindness affecting 60 million people worldwide. The optineurin (OPTN) E50K mutation was first identified in familial primary open-angle glaucoma (POAG), the onset of which is not associated with intraocular pressure (IOP) elevation, and is classified as normal-tension glaucoma (NTG). Optineurin (OPTN) is a multifunctional protein and its mutations are associated with neurodegenerative diseases such as POAG and amyotrophic lateral sclerosis (ALS). We have previously described an E50K mutation-carrying transgenic (E50K-tg) mouse that exhibited glaucomatous phenotypes of decreased retinal ganglion cells (RGCs) and surrounding cell death at normal IOP. Further phenotypic analysis of these mice revealed persistent reactive gliosis and E50K mutant protein deposits in the outer plexiform layer (OPL). Over-expression of E50K in HEK293 cells indicated accumulation of insoluble OPTN in the endoplasmic reticulum (ER). This phenomenon was consistent with the results seen in neurons derived from induced pluripotent stem cells (iPSCs) from E50K mutation-carrying NTG patients. The E50K mutant strongly interacted with TANK-binding kinase 1 (TBK1), which prohibited the proper oligomerization and solubility of OPTN, both of which are important for OPTN intracellular transition. Treatment with a TBK1 inhibitor, BX795, abrogated the aberrant insolubility of the E50K mutant. Here, we delineated the intracellular dynamics of the endogenous E50K mutant protein for the first time and demonstrated how this mutation causes OPTN insolubility, in association with TBK1, to evoke POAG.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

Structure, function and regulation of Transcription Factor IIIA: From Xenopus to Arabidopsis.

Transcription Factor IIIA (TFIIIA) is specifically required for transcription of 5S ribosomal RNA, an essential component of the ribosome. The TFIIIA protein, found in every organism, has been characterized in several species. It shows remarkably poor conservation of primary protein sequence, but all orthologues analyzed carry several C2H2-zinc fingers that are required for TFIIIA binding to both 5S ribosomal DNA (rDNA) and RNA (rRNA). Alignments of TFIIIA protein and 5S rRNA gene sequences suggest a parallel evolution of the transcription factor and its natural binding site, the internal control region of the 5S rRNA gene. We discuss here how TFIIIA expression and availability in the cell is tightly regulated at the transcriptional, post-transcriptional and post-translational level to ensure adequate amounts of TFIIIA protein depending on cell type and developmental stage. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).

Cellular and molecular biology of optineurin.

Optineurin is a gene linked to glaucoma, amyotrophic lateral sclerosis, other neurodegenerative diseases, and Paget's disease of bone. This review describes the characteristics of optineurin and summarizes the cellular and molecular biology investigations conducted so far on optineurin. Data from a number of laboratories indicate that optineurin is a cytosolic protein containing 577 amino acid residues. Interacting with proteins such as myosin VI, Rab8, huntingtin, transferrin receptor, and TANK-binding kinase 1, optineurin is involved in basic cellular functions including protein trafficking, maintenance of the Golgi apparatus, as well as NF-κB pathway, antiviral, and antibacteria signaling. Mutation or alteration of homeostasis of optineurin (such as overexpression or knockdown) results in adverse consequences in the cells, leading to the development of neurodegenerative diseases including glaucoma.

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.

Eukaryotic 5S rRNA biogenesis.

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function.

Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth.

Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.

Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation.

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.