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.

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.

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.

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.

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.

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.

Mutations of optineurin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

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.

Mechanisms of aurothiomalate-Cys2His2 zinc finger interactions.

Zinc finger motifs are present in a wide variety of regulatory proteins and generally function as interaction modules between macromolecules. These functional interactions are controlled by mechanisms of zinc (Zn2+)-binding and release. Besides Zn2+ certain electrophilic metals can potentially react with zinc finger domains and lead to changes in the structure and function of those domains. In these studies, the Cys2His2 zinc finger was chosen as a model for understanding how the gold (I) (Au1+) drug, aurothiomalate (AuTM), interacts mechanistically with the Zn2+ coordination sphere. DNA binding assays were used to analyze functional interactions between AuTM and two model Cys2His2 zinc finger transcription factors, TFIIIA and Sp1; inhibition in the micromolar range of AuTM was observed in both cases. Electrospray ionization mass spectrometry (ESI-MS) was utilized to examine molecular interactions between AuTM and a zinc finger peptide modeled after the third finger of Sp1 (Sp1-3). These experiments demonstrated Au1+ ions can bind the zinc finger structure and trigger the release of the Zn2+ ion. Quantifying the ESI-MS data allowed for a relative affinity value between Zn2+ and Au1+ ions to be calculated and shows Au1+ has a 4-fold higher affinity for Sp1-3 than Zn2+. Mechanistic differences between Zn2+ and Au1+ binding to the model Sp1-3 zinc finger were analyzed at isotopic resolution, and the metal-coordination spheres were probed with small molecules (H+, hydrogen peroxide, glutathione disulfide, and iodoacetamide). Natural isotope cluster analysis suggested the presence of a metal-thiol bond in the Cys2His2 zinc finger structure. Metal exchange reactions between zinc fingers demonstrated Zn2+ ions exchanged more rapidly than Au1+ ions. Circular dichroism (CD) exhibited differences in the secondary structure of the Sp1-3 model peptide when binding Zn2+ or Au1+ ions.

Site selection in tandem arrays of metal-binding domains.

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical TFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(II) was found to be 15 +/- 10 microM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(II) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.

Interaction of Cd2+ with Zn finger 3 of transcription factor IIIA: structures and binding to cognate DNA.

Finger 3 of transcription factor IIIA of Xenopus laevis was synthesized and constituted with Zn(2+) or Cd(2+). The C-block element of the internal control region of the promoter of the 5S rRNA gene binds to the Zn-F3 and Cd-F3 with dissociation constants of 2.6 x 10(-5) and 1.5 x 10(-4) M, respectively. According to NMR spectroscopy, Zn-F3, as well as Cd-F3, exists as a conformational equilibrium that is not susceptible to structural analysis by NMR methods. To restrict the observed conformational flexibility, a mutant F3 (mF3), which differs from F3 in the number and type of amino acids between the cysteine and the histidine ligands, was synthesized. The affinity of Zn-mF3 for the C-block DNA was greatly reduced relative to Zn-F3. Nevertheless, the metal ion dissociation constants of the Zn- and Cd-mF3 complexes remain similar to those of the native structures at 4.5 x 10(-9) and 3.2 x 10(-8) M, respectively. Zn-mF3 is more thermally stable than Cd-mF3, but both adopt similar conformations according to two-dimensional (1)H NMR spectroscopy. Each peptide displays a betabetaalpha fold for its backbone that is typical of this class of zinc finger domains. The(113)Cd ion in (113)Cd-mF3 is coupled to the protons of two cysteine and two histidine residues and characterized by a chemical shift of 567 ppm.

Cobalt-substituted zinc finger 3 of transcription factor IIIA: interactions with cognate DNA detected by (31)P ENDOR spectroscopy.

We show the first ENDOR study of the coordination environment of high-spin Co(II) in a biological system with a study of DNA binding to the Co-substituted Cys2/His2 single Zn-finger domain, Finger 3 (F3), from the prototypical zinc finger protein, transcription factor IIIA (TFIIIA) from Xenopus laevis. High covalency to cysteine and histidine is implied by ENDOR-derived 1H couplings to protons of cysteinyl ligands and 14N couplings to histidyl nitrogens, results which support the expectation that Zn(II) and Co(II) bind to F3 in a very similar manner. No changes in either 1H or 14N ENDOR were detected upon binding Co(II)-F3 to C-block DNA. Of particular importance to the use of Co(II) substitution for Zn(II), the ENDOR method shows that Co(II)-F3 undergoes sequence-specific binding to the cognate DNA for Zn(II)-F3, the internal control region (ICR) of the 5S rRNA (C-block). 31P ENDOR measurements yield a Co-31P distance of rCo-P = 8.1(3) A to the nearest backbone phosphodiester of the C-block. Interestingly, a 31P ENDOR doublet observed for Co(II)-F3 in phosphate buffer indicates that inorganic phosphate (Pi) binds at a comparable distance from Co as does the nearest phosphate of DNA, presumably at the same site.

Having it both ways: transcription factors that bind DNA and RNA.

Multifunctional proteins challenge the conventional 'one protein-one function' paradigm. Here we note apparent multifunctional proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors that bind double-stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA-binding proteins. Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT-1, TRA-1, bicoid, the bacterial sigma(70) subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle. Little is known concerning the biological roles of these alternative protein-nucleic acid interactions, and even less is known concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us to identify artificial RNA sequences that competitively inhibit a DNA-binding transcription factor not known to have a natural RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA-binding proteins is more common than currently appreciated.