Qingdu Liu

Disabling Receptor Ensembles with Rationally Designed Interface Peptidomimetics

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo- and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-induced interactions of erbB3 with different erbB receptors. A dose-dependent inhibition of native erbB receptor dimerization by the erbB peptides has been observed in 32D cell lines transfected with different combinations of erbB receptors. The peptides effectively inhibited growth of two types of transformed cells overexpressing different erbB receptors, T6-17 and 32D, in standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell viability assays. The study identifies distinct loops within the membrane-proximal part of the subdomain IV as potential receptor-receptor interaction sites for the erbB receptors and demonstrates the possibility of disabling receptor activity by structure-based targeting of the dimerization interfaces. Molecular models for possible arrangement of the erbB1·EGF complex, consistent with the involvement of subdomain IV in inter-receptor interactions, are proposed. Small dimerization inhibitors described herein can be useful as probes to elucidate different erbB signaling pathways and may be developed as therapeutic agents.

The Centrosome in Normal and Transformed Cells

The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.

Centrosomal proteins Nde1 and Su48 form a complex regulated by phosphorylation

The centrosome modulates spindle formation and plays a critical role in guiding proper segregation of chromosomes during cell division. Centrosome aberrations, frequently seen in human tumors, may cause abnormal chromosome segregation and contribute to malignant transformation. To explore the components of the centrosomes, we previously identified a novel centrosomal protein called Su48. To further characterize the Su48-containing protein ensemble in the centrosome, we performed yeast two-hybrid screens and isolated a number of Su48-interacting molecules, including the centrosomal protein Nde1. Here, we demonstrate that Su48 can associate with Nde1. Moreover, we found that Nde1 is subjected to phosphorylation in vivo. In particular, we identified six putative Cdc2 phosphorylation sites in Nde1 and found that alteration of these sites diminishes phosphorylation by Cdc2 in vitro and affects the stability of Su48-Nde1 interactions and the centrosomal localization of Nde1. Ablation of Nde1 by gene specific small interfering RNA causes mitotic delay and cell death, coupled with a modest decrease in the incidence of the cells that harbor excessive centrosomes. Collectively, our findings indicate that Nde1 can form a protein complex with Su48 in the centrosome and plays an important role for successful mitosis.

p78/MCRS1 forms a complex with centrosomal protein Nde1 and is essential for cell viability.

The centrosome, an organelle that functions as the major microtubule-organizing center, plays an essential role in the formation of the mitotic spindle and guiding accurate chromosome segregation. Centrosome aberrations are frequently associated with various forms of human cancers and it is thought that defects in this organelle contribute to genomic instability and malignant transformation. We recently identified and characterized a centrosome-localized protein complex that is comprised of Su48 and Nde1. Disruption of the normal function of these proteins leads to abnormal cell division. To extend our understanding of how this protein complex operates, we sought to identify Nde1-interacting molecules by the yeast two-hybrid screening method. Here, we demonstrate that both Nde1 and Su48 can associate with p78/MCRS1, a protein implicated in cancer development. We found that, whereas the majority of p78 localizes to the nucleus as reported in earlier studies, a fraction of the p78 protein can be detected in the centrosome. Moreover, we determined that a region containing the forkhead-associated domain of p78 is involved in association with Nde1 and Su48, as well as in centrosomal localization. We also provide evidence that the association between p78 and Nde1 is regulated by phosphorylation on Nde1. Furthermore, abrogation of the endogenous p78 function by small interfering RNA knockdown causes cell death and a modest delay in mitosis. These results indicate that a subset of the p78 proteins comprises a component of the centrosome and that p78 is essential for cell viability.

Nek2 targets the mitotic checkpoint proteins Mad2 and Cdc20: A mechanism for aneuploidy in cancer

In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2-Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.