Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms

Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.     

A model of troponin-I in complex with troponin-C using hybrid experimental data: the inhibitory region is a beta-hairpin

We present a model for the skeletal muscle troponin-C (TnC)/troponin-I (TnI) interaction, a critical molecular switch that is responsible for calcium-dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin-C in complex with troponin-I, or in the ternary troponin-complex, have not yet delivered a high-resolution structure. Many groups have pursued different experimental strategies, such as X-ray crystallography, NMR, small-angle scattering, chemical cross-linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left-handed sense (Model L), and another that winds about TnC in a right-handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross-linking and FRET data. The inhibitory region TnI(95-114) is modeled as a flexible beta-hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a beta-hairpin region of the actin-binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the beta-hairpin is an important structural motif that communicates the Ca2+-activated troponin regulatory signal to actin.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

三叶木通花粉前纤维蛋白基因的克隆与表达

以三叶木通花蕾为材料,采用RT-PCR、3-′RACE方法克隆了三叶木通花粉前纤维蛋白基因,命名为Atf-Pro(GenBank登录号GQ478584)。结果表明:AtfPro的cDNA全长735 bp、阅读框393 bp、编码131个氨基酸,有1个342 bp的3′端非翻译区。预测分子量约为14.081 kD,等电点4.74。氨基酸和核苷酸序列的同源性分析发现,AtfPro基因属于植物花粉profilin基因家族的新成员。RT-PCR定性分析表明,AtfPro基因在三叶木通花蕾、花药、雌花花瓣和柱头组织中均有表达,但在幼叶、茎尖、根尖组织中低水平表达或不表达,生殖器官中的表达时期从花序分化发育开始到开花散粉结束。     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

Profilin1 facilitates staurosporine-triggered apoptosis by stabilizing the integrin β1-actin complex in breast cancer cells

Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5β1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5β1. These results suggest that the insufficient fibronectin caused by the integrin α5β1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin β1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5β1, and suggested a new target for breast cancer therapy.     

Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA

In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.     

水稻花粉[肌动蛋白]抑制蛋白基因的克隆和表达分析

[肌动蛋白]抑制蛋白（profilin）是一种低分子量、与肌动蛋白结构的蛋白质。通过筛选水稻成熟花粉的cDNA文库,获得了两个全长cDNA片段,序列分析结果表明,两个cDNA片段长度分别为821bp和805bp;共同拥有一个由131个氨基酸组成的开放密码框、5′末端翻译区和一个带有poly(A)的3′区域。[肌动蛋白]抑制蛋白与玉米、C.dactylon、H.brasiliensis、P.pratense中的该蛋白质的同源性分别为89％、87％、83％、89％。Southern杂交分析显示,在基因组至少有两个基因存在。Northern杂交和RT-PCR结果显示它在花粉和花粉中特异表达。     

Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43

Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition.