Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein

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Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).     

Comparison of methods of repelling mosquitoes

Comparisons were made with human subjects of repellency of free flying Anopheles gambiae using soaps containing deet and permethrin or permethrin alone. The reduction in biting did not differ statistically significantly between these two forms of repellent soap. Some mosquitoes which touched the permethrin soap layer without biting died within the next day. The soap with deet was compared with the same amount of deet in a commercially available stick or as a liquid. Only in the latter form did the repellent show resistance to being washed off by cold water.Electrically heated vaporizing mats containing bio-allethrin were shown to be more effective than smouldering coils containing natural pyrethrins, in that there was more complete prevention of biting, and more rapid knock-down and kill. Operation of the vaporizing mat caused deposition of insecticide on the room surfaces.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.     

Predicting oligomerization states of coiled coils.

An algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two- and three-stranded, coiled-coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations--profile values of solvent-exposed residues and of amino acids that were underrepresented in the data-base were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.     

Interlock可解脱弹簧圈在脾动脉瘤腔内治疗中的应用

目的:探究Interlock可解脱弹簧圈在脾动脉瘤的腔内治疗中的应用价值。方法:回顾性分析2016年2月至2019年2月于本中心使用Interlock可解脱弹簧圈治疗的36例脾动脉瘤患者的临床资料,包括10例男性,26例女性,31例真性动脉瘤,5例假性脾动脉瘤,术前均行超声或CTA明确诊断。术中栓塞后,立即血管造影以明确技术成功率。围手术期及术后2周、3个月和6个月监测血常规、胰淀粉酶和主动脉CTA,观察并发症的发生情况。结果:6例患者弹簧圈栓塞动脉瘤远近端及瘤腔,其余仅栓塞脾动脉瘤瘤腔。术中DSA血管造影提示即刻脾动脉瘤栓塞闭塞率为97%以上,术中共使用128枚Interlock弹簧圈,其中104枚为钻石型,24枚为普通2D型。弹簧圈平均直径为6.3±4.2(2-12)mm,平均长度为16±13.5(3-32)mm,瘤体平均尺寸为40.6±12.5(15-70) mm。围手术期无并发症发生。平均随访10.0±3.2(6-15)个月,1月后2例出现脾梗死,7例发生轻微腹痛及低热等症状,所有病例均未见瘤腔再通和瘤体增大。结论:Interlock可解脱弹簧圈可以安全有效地治疗脾动脉瘤,但其远期疗效需长期随访观察。     

Self‐assembly of designed coiled coil peptides studied by small‐angle X‐ray scattering and analytical ultracentrifugation

α‐Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix‐containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small‐angle X‐ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self‐assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self‐assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Rho‐associated coiled‐coil kinase 1 activation mediates amyloid precursor protein site‐specific Ser655 phosphorylation and triggers amyloid pathology

Rho‐associated coiled‐coil kinase 1 (ROCK1) is proposed to be implicated in Aβ suppression; however, the role for ROCK1 in amyloidogenic metabolism of amyloid precursor protein (APP) to produce Aβ was unknown. In the present study, we showed that ROCK1 kinase activity and its APP binding were enhanced in AD brain, resulting in increased β‐secretase cleavage of APP. Furthermore, we firstly confirmed that APP served as a substrate for ROCK1 and its major phosphorylation site was located at Ser655. The increased level of APP Ser655 phosphorylation was observed in the brain of APP/PS1 mice and AD patients compared to controls. Moreover, blockade of APP Ser655 phosphorylation, or inhibition of ROCK1 activity with either shRNA knockdown or Y‐27632, ameliorated amyloid pathology and improved learning and memory in APP/PS1 mice. These findings suggest that activated ROCK1 targets APP Ser655 phosphorylation, which promotes amyloid processing and pathology. Inhibition of ROCK1 could be a potential therapeutic approach for AD.