3,069 research outputs found
Chaperone/usher machinery
No full textMany virulence organelles of Gram-negative bacterial pathogens are assembled via the periplasmic chaperone/usher (CU) pathway. The assembly process is a complex task, involving secretion of organelle subunits via the two membranes and periplasm, subunit folding and assembly. In this thesis, the mechanism of the organelle subunit trafficking and assembly via the CU pathway was investigated at different steps, subunit capture by the chaperone (paper II), usher targeting (paper III), and transport through the usher (paper I), using the Caf1M/Caf1M CU system that assembles the Yersinia pestis F1 capsular antigen from Caf1 subunits.
In paper II, we performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 subunit and analyzed the effect of the mutations on structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. We show that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enable Caf1M to rapidly capture/fold Caf1 subunit. The switch from the Caf1M-Caf1 contact to the Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M with improved chaperone function was engineered. In paper III, we elucidated the mechanism of the usher-targeting step at atomic resolution. We show that a pair of conserved proline residues in free chaperone forms a ‘proline lock’, which blocks the usher binding. Binding of subunit to the chaperone opens the proline lock and allows the chaperone-subunit complex to bind to the usher. We show that this proline lock exists in other CU systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of free chaperone. In paper I, a novel middle domain of Caf1A usher (UMD) was isolated and its crystal structure was determined. We show that UMD and Caf1 fibre subunit displayed significant structural similarity. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1-fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher
Characterization of the Usher Syndrome gene CDH23: implications for mechanosensation in the vertebrate inner ear
No full textDeafness is the most common form of sensory impairment afflicting the human population. Approximately one in eight hundred children is born with serious hearing impairment and more than half of these cases are likely due to single gene defects. In addition to hearing loss, mutations in some genes cause Usher Syndrome, not only affecting the auditory apparatus but also causing visual impairment eventually leading to blindness. Several genetic loci have been linked to Usher Syndrome Type I, the most severe form of the disease, and so far five of the relevant genes have been identified. Understanding their molecular role in the context of ear and retina physiology will be invaluable to the design of effective therapies against this devastating disease. Some forms of Usher Syndrome as well as other hearing disorders are caused by defects in the inner ear that contains the end organs for the perception of sound waves, the cochlea, and for the detection of gravity and acceleration, the vestibule. Both end organs contain mechanosensory hair cells that are named after actin rich stereocilia projecting from their apical surface. The stereocilia contain mechanically gated ion channels that open or close upon deflection of the stereocilia. This in turn triggers ion influx into the hair cells, causing changes in cell polarization and alterations in the rate of neurotransmitter release from the hair cells onto innervating neurons. The mechanically gated transduction channel implicated in this event has remained elusive. Mechanical gating of the transduction channel is believed to be triggered by thin filaments, the tip links, connecting adjacent stereocilia into a bundle. It has been suggested that these connector molecules are being stretched during hair cell stimulation, thereby actively pulling open the transduction channel. Although these filaments are clearly detectable on the ultrastructural level, their molecular nature has remained elusive. One molecule that might participate in mechanoelectrical transduction is the transmembrane protein cadherin 23 (CDH23). Mutations in its gene can cause Usher Syndrome, non-syndromic forms deafness and age-related hearing loss in human patients. Mice and zebrafish that carry mutations in the orthologous genes show splayed stereocilia bundle morphology, arguing for a function of the protein product in the cell compartment harboring the transduction channel. Furthermore, CDH23 is large enough to be the tip link, the extracellular filament proposed to gate the mechanotransduction channel.
Here we show that antibodies against CDH23 label the entire stereocilia bundle during hair cell morphogenesis. In mature hair cells CDH23 labelling is confined to the tip links. Further, CDH23 has biochemical properties similar to those of the tip link. In cell-aggregation experiments CDH23 displays Ca2+-dependent, homophilic adhesion potential, an attribute typically observed for members of the cadherin superfamily, which may explain how adjacent stereocilia are linked together. Moreover, CDH23 forms a complex with myosin 1c (MYO1C), the only known component of the mechanotransduction apparatus, suggesting that CDH23 and MYO1C cooperate to regulate the activity of mechanically gated ion channels in hair cells.
Computer assisted alignments with sequences encoding the cytosolic domain of CDH23 reveal two putative PDZ-binding motifs. Others and we can show that CDH23 interacts with the product of a second Usher Syndrome gene, harmonin. Two PDZ domains within harmonin interact with two complementary binding surfaces in the CDH23 cytoplasmic domain. One of the binding surfaces is disrupted by sequences encoded by an alternatively spliced CDH23 exon that is expressed in hair cells, but not in any other tissue analyzed so far. In the ear, harmonin is expressed in the stereocilia of developing hair cells. Since mice with a targeted deletion of the harmonin gene have been reported to phenocopy the splayed stereocilia bundle morphology observed in CDH23 deficient mice, the complex of the two Usher Syndrome proteins is predicted to be important for the stereocilia bundle. Whether the harmonin–CDH23 complex might be involved in mechanotransduction is unclear, since harmonin´s presence in mature stereocilia has not been reported yet.
We concluded that CDH23 may serve a dual function in auditory hair cells: together with harmonin the molecule is important to shape the hair bundle during hair bundle morphogenesis and in mature stereocilia the molecule is part of the tip link complex
Usher syndrome associated with Fuchs’ heterochromic uveitis: a case report
No full textEce Turan-Vural, Banu Torun-Acar, Nejla Tükenmez, M Şahin Sevim, Bulent Buttanri, Suphi AcarOphthalmology Clinic, Haydarpasa Numune Education and Research Hospital, Istanbul, TurkeyAbstract: We report a case of Usher syndrome in association with unilateral Fuchs' heterochromic uveitis.Keywords: Fuchs’ heterochromic uveitis, Usher syndrome, deafness, blindnes
Development of a genotyping microarray for Usher syndrome
No full textBACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool
Comment on Ryder's SINBAD Neurosemantics: Is Teleofunction Isomorphism the Way to Understand Representations?
No full textThe merit of the SINBAD model is to provide an explicit mechanism showing how the cortex may come to develop detectors responding to correlated properties and therefore corresponding to the sources of these correlations. Here I argue that, contrary to the article, SINBAD neurosemantics does not need to rely on teleofunctions to solve the problem of misrepresentation. A number of difficulties for the teleofunction theories of content are reviewed and an alternative theory based on categorization performance and statistical relations is argued to provide a better account and to come closer to the practice in neuroscience and to powerful intuitions on swampkinds and on broad/narrow content
Letter from Office of Representative Usher Burdick to Floyd Montclair Regarding Per Capita Payments, November 12, 1951
No full textThis letter, dated November 12, 1951, from the office of United States (US) Representative Usher Burdick to Three Affiliated Tribes Member Floyd Montclair concerns per capita payments. The author thanks Montclair for his letter of November 9 and an enclosed resolution by the Three Affiliated Tribes of the Fort Berthold Reservation, and assures Montclair that the message has been forwarded to Burdick, who is currently in Williston.https://commons.und.edu/burdick-papers/1051/thumbnail.jp
President Lincoln\u27s cabinet, by Honorable John P. Usher, secretary of the interior January 7, l863-May 15, l865, with a foreword and a sketch of the life of the author by Nelson H. Loomis ...
No full textThe pamphlet includes a brief biography of the author, John Palmer Usher. The original remarks concerning President Lincoln\u27s Cabinet were made during a speech delivered at a banquet given by Mr. D. M. Edgerton in honor of Judge D. D. Hoag in Wyandotte, Kansas on June 20, 1887. The cover of the pamphlet includes the full title with a four line boarder surrounding the title.https://scholarsjunction.msstate.edu/fvw-pamphlets/2068/thumbnail.jp
Moderne Arbeitsmethoden im Maschinenbau
No full textVon John T. Usher ; Autorisirte deutsche Bearbeitung von A. ElfesExlibrisstempel: "Schweizerische Bauzeitung Redaction A. Waldner, Ing. Zürich" 002194263_0001 Exemplar der ETH-BIBGeschenkexlibris-Etikette: "Geschenk aus dem Nachlass von August Waldner" 002230265_0001 Exemplar der ETH-BI
Crystal structure of the P pilus rod subunit PapA
No full textP pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G(1) beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it
Correspondence Between Mrs. B. Usher & Family and Students at B. T. W. High School, 1931
No full textCorrespondence between Mrs. B. Usher & Family and Students at B. T. W. High School including a hank you letter from the students at Booker T. Washington High School to [Bazoline E. Usher]. 1 page.The Atlanta University Center Robert W. Woodruff Library acknowledges the generosity of the Digital Public Library of America for supporting in part the digitization of this collection as part of the Black Women's Suffrage Digital Collection, a project made possible through funding from Pivotal Ventures, A Melinda Gates Company
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