Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1) in nasopharyngeal epithelial cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

The kinetics of p53 activation versus cyclin E accumulation underlies the relationship between the spindle-assembly checkpoint and the postmitotic checkpoint

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21(CIP1/WAF1) were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21(CIP1/WAF1), is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21(CIP1/WAF1). These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.     

Loss of ΔNp63α promotes mitotic exit in epithelial cells

Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.