A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer

Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein–dynactin and Mad1–Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.

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Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses

Microorganisms ◽

10.3390/microorganisms8081207 ◽

2020 ◽

Vol 8 (8) ◽

pp. 1207

Author(s):

Fabian Z. X. Lean ◽

Jean Payne ◽

Jennifer Harper ◽

Joanne Devlin ◽

David T. Williams ◽

...

Keyword(s):

Infected Cell ◽

Bluetongue Virus ◽

Cross Reactivity ◽

Infected Sheep ◽

Immunohistochemical Detection ◽

Structural Protein ◽

Diverse Range ◽

Serotype 1 ◽

Development And Validation ◽

Formalin Fixed

The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.

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Fine structure and molecular organization of the periostracum in a gastropod mollusc Buccinum undatum L. and its relation to similar structural protein systems in other invertebrates

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1978.0048 ◽

1978 ◽

Vol 283 (998) ◽

pp. 417-459 ◽

Cited By ~ 23

Keyword(s):

Electron Microscopy ◽

Cholesteric Liquid Crystal ◽

Three Dimensional ◽

Coiled Coil ◽

Longitudinal Axis ◽

Molecular Organization ◽

Structural Protein ◽

Buccinum Undatum ◽

Basic Features ◽

Smooth Transitions

The horny layer of periostracum which covers the shell of Buccinum undatum L. has been studied by a combination of the fine structural techniques including high resolution transmission electron microscopy and scanning electron microscopy as well as by chemical analysis and X-ray diffraction. It has been found that the main structural component is a tectin type protein with globular and probably coiled-coil α-helical regions accompanied by a small amount of polysaccharide. Much of the periostracum is built up of protein sheets superposed in a regular manner and stabilized by some type of covalent cross-linking involving aromatic molecules. The protein is one of the class of structural macromolecules called scleroproteins. Each sheet of protein is made up of molecular sub-units which have a characteristic dumb-bell shape and which are about 32 nm long and 6.5 nm wide at their globular ends. End-to-end long-axis aggregation of these units produces filaments which aggregate further by side-to-side association into ribbons and ultimately sheets. The side-to-side association is always in register and hence the sheets have a major transverse striation repeating at 32 nm intervals. The protein sheets can be ascribed a longitudinal axis in terms of the direction of their component filaments. On this basis it can be shown that successive superposed sheets are rotated in a horizontal plane through an angle of 20-25° relative to one another, in a constant direction either clockwise or anticlockwise. Such helicoidal organization is of the cholesteric liquid crystal type which is often found in a biological context, e.g. chitin fibril disposition in arthropod cuticle. This helicoidal layering of the protein sheets is manifested in oblique sections of periostracum as repeated parabolic lamellae. Irregularities in the form of the parabolic lamellae can be accounted for on the basis of the curvature and extensive folding of the periostracum. The outer and innermost layers of the periostracum tend not to show helicoidal organization but exhibit a different aggregation mode of the dumb-bell-shaped units into a three-dimensional hexagonally packed network matrix. This matrix is much interrupted by vacuoles and localized smooth transitions into the ribbon mode of aggregation. This ability to exist in both fibrous and network aggregation states is comparable to that known among the collagens and muscle proteins. The amino acid compositions and conformations of proteins which can form cholesteric helicoidal systems are reviewed and compared with the protein of Buccinum periostracum. This property is apparently confined to alpha helical rod-shaped proteins and globular tektins. The beta conformation does not favour cholesteric organization. The structures and compositions of other molluscan periostraca and periostracum- like structures from other invertebrate phyla are compared with the periostracum of Buccinum . While all periostraca and functionally related structures have certain basic features in common there is a considerable degree of variation at the molecular and organizational levels.

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