Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.
While the majority of host cell protein (HCP) impurities are effectively removed in typical downstream purification processes, a small population of HCPs are particularly challenging. Previous studies have identified HCPs that are challenging for a variety of reasons. Lipoprotein lipase (LPL) – a Chinese hamster ovary (CHO) HCP that functions to hydrolyze esters in triglycerides – was one of ten HCPs identified in previous studies as being susceptible to retention in downstream processing. LPL may degrade polysorbate 80 (PS-80) and polysorbate 20 (PS-20) in final product formulations due to the structural similarity between polysorbates and triglycerides. In this work, recombinant LPL was found to have enzymatic activity against PS-80 and PS-20 in a range of solution conditions that are typical of mAb formulations. LPL knockout CHO cells were created with CRISPR and TALEN technologies and resulting cell culture harvest fluid demonstrated a significantly reduced polysorbate degradation without significant impact on cell viability when compared to wild type samples.
Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead. ReceiVed December 5, 2006; ReVised Manuscript ReceiVed March 7, 2007 ABSTRACT: Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in Vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in Vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.Protein kinases have evolved a variety of regulatory mechanisms that exploit the effects of site-specific phosphorylation and precise protein/domain interactions (1, 2). The largest family of nonreceptor protein tyrosine kinases consists of the eleven Src kinases (3) that each contain an N-terminal unique region, followed by an Src homology-3 (SH3 1 ) domain, Src homology-2 (SH2) domain, catalytic domain, and a C-terminal tail with a conserved regulatory tyrosine. A significant leap in our understanding of kinase regulation has been provided by the three-dimensional structures of two Src family kinases (Hck and Src) in their inactive states (4-6). These structures reveal that the SH3 and SH2 domains are located on the "back" surface of the kinase domain mediating a network of intramolecular interactions th...
A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm. By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.
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