Insights into the Function of the CRM1 Cofactor RanBP3 from the Structure of Its Ran-Binding Domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins

Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment

Self-Organization of Anastral Spindles by Synergy of Dynamic Instability, Autocatalytic Microtubule Production, and a Spatial Signaling Gradient

Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient

The feasibility of gene therapy in the treatment of head and neck cancer

Standard approach to the treatment of head and neck cancer include surgery, chemotherapy, and radiation. More recently, dramatic increases in our knowledge of the molecular and genetic basis of cancer combined with advances in technology have resulted in novel molecular therapies for this disease. In particular, gene therapy, which involves the transfer of genetic material to cells to produce a therapeutic effect, has become a promising approach. Clinical trials concerning gene therapy strategies in head and neck cancer as well as combination of these strategies with chemotherapy and radiation therapy will be discussed

High prevalence of vitamin D deficiency among children aged 1 month to 16 years in Hangzhou, China

<p>Abstract</p> <p>Background</p> <p>Recent studies have suggested that vitamin D deficiency in children is widespread. But the vitamin D status of Chinese children is seldom investigated. The objective of the present study was to survey the serum levels of 25-hydroxyvitamin D [25(OH)D] in more than 6,000 children aged 1 month to 16 years in Hangzhou (latitude: 30°N), the capital of Zhejiang Province, southeast China.</p> <p>Methods</p> <p>The children aged 1 month to 16 years who came to the child health care department of our hospital, the children's hospital affiliated to Zhejiang university school of medicine, for health examination were taken blood for 25(OH) D measurement. Serum 25(OH) D levels were determined by direct enzyme-linked immunosorbent assay and categorized as < 25, < 50, and < 75 nmol/L.</p> <p>Results</p> <p>A total of 6,008 children aged 1 month to 16 years participated in this cross-sectional study. All the subjects were divided into subgroups according to their age: 0-1y, 2-5y, 6-11y and 12-16y representing infancy, preschool, school age and adolescence stages respectively. The highest mean level of serum 25(OH)D was found in the 0-1y stage (99 nmol/L) and the lowest one was found in 12-16y stage (52 nmol/L). Accordingly, the prevalence of serum 25(OH)D levels of < 75 nmol/L and < 50 nmol/L were at the lowest among infants (33.6% and 5.4% respectively) and rose to the highest among adolescents (89.6% and 46.4% respectively). The mean levels of serum 25(OH)D and the prevalence of vitamin D deficiency changed according to seasons. In winter and spring, more than 50% of school age children and adolescents had a 25(OH)D level at < 50 nmol/L. If the threshold is changed to < 75 nmol/L, all of the adolescents (100%) had low 25(OH)D levels in winter and 93.7% school age children as well.</p> <p>Conclusions</p> <p>The prevalence of vitamin D deficiency and insufficiency among children in Hangzhou Zhejiang province is high, especially among children aged 6-16 years. We suggest that the recommendation for vitamin D supplementation in Chinese children should be extended to adolescence.</p

Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology

YesThe contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner.This work was supported by SFB 670 and DFG NO 113/22. K.B. was supported by a fellowship from the NRW International Graduate School “From Embryo to Old Age: the Cell Biology and Genetics of Health and Disease” (IGSDHD), Cologne

Nucleo-cytoplasmic transport of proteins and RNA in plants

Merkle T. Nucleo-cytoplasmic transport of proteins and RNA in plants. Plant Cell Reports. 2011;30(2):153-176.Transport of macromolecules between the nucleus and the cytoplasm is an essential necessity in eukaryotic cells, since the nuclear envelope separates transcription from translation. In the past few years, an increasing number of components of the plant nuclear transport machinery have been characterised. This progress, although far from being completed, confirmed that the general characteristics of nuclear transport are conserved between plants and other organisms. However, plant-specific components were also identified. Interestingly, several mutants in genes encoding components of the plant nuclear transport machinery were investigated, revealing differential sensitivity of plant-specific pathways to impaired nuclear transport. These findings attracted attention towards plant-specific cargoes that are transported over the nuclear envelope, unravelling connections between nuclear transport and components of signalling and developmental pathways. The current state of research in plants is summarised in comparison to yeast and vertebrate systems, and special emphasis is given to plant nuclear transport mutants

Combinatory cytotoxic effects produced by E1B-55kDa-deleted adenoviruses and chemotherapeutic agents are dependent on the agents in esophageal carcinoma

We examined possible combinatory antitumor effects of replication-competent type 5 adenoviruses (Ad) lacking E1B-55kDa molecules (Ad-delE1B55) and chemotherapeutic agents in nine human esophageal carcinoma cells. Ad-delE1B55 produced cytotoxic effects on all the carcinoma cells and the cytotoxicity is not directly linked with the p53 status of the tumors or with the infectivity to respective tumors. A combinatory treatment with Ad-delE1B55 and an anticancer agent, 5-fluorouracil (5-FU), mitomycin C or etoposide, produced greater cytotoxic effects than that with either the Ad or the agent. Administration of 5-FU could minimally inhibit the viral replication and a simultaneous treatment with the Ad and 5-FU achieved better cytotoxicity than sequential treatments. We also confirmed the antitumor effects by the combination of Ad-delE1B55 with 5-FU in vivo. Cisplatin, however, did not achieve the combinatory effects in most of the cells tested. These data indicate that the Ad-delE1B55 produce combinatory antitumor effects with a chemotherapeutic agent irrespective of the administration schedule, but the effects depend on an agent in esophageal carcinoma

Factors Contributing to the Biofilm-Deficient Phenotype of Staphylococcus aureus sarA Mutants

Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin) that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases). Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results demonstrate that the inability of a sarA mutant to repress production of extracellular nuclease and multiple proteases have independent but cumulative effects that make a significant contribution to the biofilm-deficient phenotype of an S. aureus sarA mutant