Wendy Sandoval

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Microwave Assisted Proteomics

State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instrumentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characterization workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectrometric characterization. Techniques for microwave-assisted bio-catalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of post-translational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied.

Ubiquitin ligase RNF146 regulates tankyrase and Axin to promote Wnt signaling.

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse

Mia3’s contribution to protein secretion is broader than previously realized—its absence impairs collagen deposition and normal development of cartilage and bone.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn’s disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7.

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

Host cell protein testing by ELISAs and the use of orthogonal methods

Host cell proteins (HCPs) are among the process‐related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process‐related impurities are needed to support process development, process validation, and control system testing. A multi‐analyte enzyme‐linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti‐HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non‐immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution‐dependent non‐linearity with multi‐product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co‐purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low‐level HCPs is also outlined, with consideration of clinical information. Biotechnol. Bioeng. 2014;111: 2367–2379.

mTORC1-Dependent Metabolic Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells.

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies.1,2 The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.