• White individuals have a high frequency of the common PAR4 gene (F2RL3) variant Ala120; blacks have a high frequency of Thr120.• PAR4 Thr120 induces greater signaling and is associated with greater platelet aggregation and reduced inhibition by a PAR4 antagonist.Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n 5 70) express 14% more PAR4 protein than those from whites (n 5 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca 21 flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. (Blood. 2014;124(23):3450-3458)

SUMMARY V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high fidelity, classical non-homologous end joining (cNHEJ) machinery. Previous studies implicated the RAG/DNA post-cleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here we report that RAG2’s “acidic hinge”, previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

Current theories suggest that mitotic checkpoint proteins are essential for proper cellular response to taxanes, a widely-used family of chemotherapeutic compounds. We recently demonstrated that absence or depletion of protein Daxx increases cellular taxol (paclitaxel) resistance—a common trait of patients diagnosed with several malignancies, including breast cancer. Further investigation of Daxx-mediated taxol response revealed that Daxx is important for the proper timing of mitosis progression and cyclin B stability. Daxx interacts with mitotic checkpoint protein Rassf1 and partially co-localizes with this protein during mitosis. Rassf1/Daxx depletion or expression of Daxx binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance in cells and mouse xenograft models. In breast cancer patients, we observed the inverse correlation between Daxx and clinical response to taxane-based chemotherapy. These data suggest that Daxx and Rassf1 define a mitotic stress checkpoint that enables cells to exit mitosis as micronucleated cells (and eventually die) when encountered with specific mitotic stress stimuli, including taxol. Surprisingly, depletion of Daxx or Rassf1 does not change activity of E3 ubiquitin ligase APC/C in in vitro settings, suggesting necessity of mitotic cellular environment for proper activation of this checkpoint. Daxx and Rassf1 may become useful predictive markers for the proper selection of patients for taxane chemotherapy.

Resistance to the anti-neoplastic drug paclitaxel is frequent in breast cancer patients. Most studies of paclitaxel resistance have focused on pathways that elicit cellular response, while little is known about players involved in the acquirement of taxane resistance. By screening a cohort of breast cancer cell lines, we observed a correlation between level of protein Daxx and response to paclitaxel. Cells lines expressing increased level of Daxx displayed a robust paclitaxel response with nearly all cells undergoing micronucleation, while cell lines with low amount of Daxx showed a decrease in micronucleation, and accumulation in mitosis. At used paclitaxel concentrations, apoptotic levels were negligible in all cell lines tested. Human cell lines expressing anti-Daxx siRNA as well as Daxx-/- mouse fibroblasts showed similar cellular response to paclitaxel. Importantly, absence or depletion of Daxx resulted in cell survival after paclitaxel treatment, as measured by colony formation assay. We conclude that Daxx may be an important predictive factor in cellular response to paclitaxel, which emphasizes a critical but unknown function of this protein in mitotic progression, which, when disabled, leads to survival advantages upon paclitaxel treatment.