For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. INTRODUCTIONAs points of structural linkage between the extracellular matrix (ECM) and the intracellular cytoskeleton, focal adhesions possess a complex function. For example, during migration, cells must rapidly break down and reform adhesions with the ECM, providing force for propulsion (Lauffenburger and Horwitz, 1996). At mitotic entry, cultured cells round up and decrease adhesion to the ECM; at mitotic exit, basal attachments reassemble and contribute to the force generation required for efficient progress through cytokinesis and reentry into G 1 . In interphase cells, the formation of novel focal adhesion-ECM interactions can specify cellular differentiation by activating specific signaling cascades culminating in the induction of differentiationpromoting transcription factors, and in parallel enforce removal from the cell cycle (Boudreau and Bissell, 1998). In many cell types, sustained loss of adhesion is a sufficient stimulus to induce apoptosis (anoikis) (Frisch and Francis, 1994), a surveillance mechanism against cancer, inhibiting the formation of micrometastases. Hence, one frequent effect of oncogenic transformation is the circumvention of the adhesion-viability coupling, leading to acquisition by cancer cells of the ability to grow in an anchorage-independent manner (Schwartz, 1997). Based on these diverse biological roles, there has been considerable research effort directed at elucidating the signaling role of focal adhesion-associated proteins (Schlaepfer et al., 1999).HEF1, p130Cas, and Efs/Sin define the Cas family of proteins Bouton et al., 2001). In interphase cells, Cas proteins predominantly localize to focal adhesions. During initial integrin engagement, induced by cell a...

Human enhancer of invasion, clone 10 (HEI10) (CCNB1IP1) was first described as a RING-finger family ubiquitin ligase that regulates cell cycle by interacting with cyclin B and promoting its degradation. Subsequently, other studies suggested specific upregulation of HEI10 in metastatic melanoma and demonstrated direct interaction between HEI10 and the tumor suppressor Merlin, encoded by the neurofibromatosis 2 gene. These and other results led us to hypothesize that HEI10 also influences the processes of cell migration and metastasis. We here show that cells with depleted HEI10 both migrate more rapidly and invade more effectively than control cells. HEI10 depletion post-transcriptionally increases the expression of a group of promotility regulatory proteins including p130Cas, paxillin, Cdk1 and cyclin B2, but excluding Merlin. Among these, only inhibition of Cdk1/ cyclin B activity specifically reversed the motility and invasion of HEI10-depleted cells. Finally, HEI10 is abundantly transcribed in many human tissues, and particularly abundant in some tumor cell lines, suggesting that it may be commonly involved in coordinating cell cycle with cell migration and invasion.

Employment of the decision strategies outlined in this general discussion should help to pinpoint mode of activity in drug development and validation. Overall, as a paradigm for drug development, a search for small molecules that can interfere with PPIs would seem to have significant long term potential. At present, the level of structural knowledge in databases is not sufficient to predict in toto the protein binding properties of a modeled drug, but as databases improve, this may become generally feasible. A major point that remains to be determined is how much specificity of protein binding can be incorporated into molecules of generally less than 500 Da. Finally, integration of PPI-targeting strategies with other approaches towards drug design will enhance the number of signaling pathways that can effectively be targeted. These points will be particularly pertinent as technologies permit a systematic identification of encoded protein interactions that govern the proteornic complement of cells.

Fibrolamellar carcinoma (FLC) of the liver is a rare variant of hepatocellular carcinoma (HCC). Here we report the case of a 12-year-old Indian male with typical FLC with no apparent hepatitis B virus (HBV) infection and a non-cirrhotic liver. The patient, though seronegative for HBsAg, showed expression of HBcAg in both the liver and tumour tissue. RT-PCR analysis revealed the presence of full-length HBx-transcripts in both liver/tumour tissue, along with truncated HBx-transcripts only in the tumour tissue. The lymphocytes in both peripheral and liver/tumour compartments showed a proliferative response to either/or HBcAg and HBxAg, which could be further augmented on addition of rIL-2. This is the first study to show not only the presence of HBcAg in the liver/tumour tissue but also prior exposure of the FLC patient's lymphocytes to HBV antigens. Also, the presence of the full-length and truncated HBx-transcripts in the tumour tissue, a proposed tumorigenic marker for hepatocarcinogenesis in chronic HBV patients, suggests an oncogenic role of HBV in this rare variant of HCC.

The role of calcium release-activated calcium (CRAC) channels is well characterized and is of particular importance in T-cell function. CRAC channels are involved in the pathogenesis of several autoimmune diseases, making it an attractive therapeutic target for treating inflammatory diseases, like rheumatoid arthritis (RA). A systematic structure–activity relationship study with the goal of optimizing lipophilicity successfully yielded two lead compounds, 36 and 37. Both compounds showed decent potency and selectivity and a remarkable pharmacokinetic profile. Further characterization in in vivo RA models and subsequent histopathological evaluation of tissues led to the identification of 36 as a clinical candidate. Compound 36 displayed an excellent safety profile and had a sufficient safety margin to qualify it for use in human testing. Oral administration of 36 in Phase 1 clinical study in healthy volunteers established favorable safety, tolerability, and good target engagement as measured by levels of IL-2 and TNF-α.