Improved Production and Biophysical Analysis of Recombinant Silicatein-α.

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon-oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.

Protein kinase Cα gain-of-function variant in Alzheimer's disease displays enhanced catalysis by a mechanism that evades down-regulation.

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca2+ and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKCα in Alzheimer's disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ∼30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca2+ or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKCα-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKCα-M489V mutation. Our results unveil how an AD-associated mutation in PKCα permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKCα.

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer.

Somatically mutated ABL1 is an actionable and essential NSCLC survival gene.

The lack of actionable mutations in patients with non-small cell lung cancer (NSCLC) presents a significant hurdle in the design of targeted therapies for this disease. Here, we identify somatically mutated ABL1 as a genetic dependency that is required to maintain NSCLC cell survival. We demonstrate that NSCLC cells with ABL1 mutations are sensitive to ABL inhibitors and we verify that the drug-induced effects on cell viability are specific to pharmacological inhibition of the ABL1 kinase. Furthermore, we confirm that imatinib suppresses lung tumor growth in vivo, specifically in lung cancer cells harboring a gain-of-function (GOF) mutation in ABL1. Consistent with structural modeling, we demonstrate that mutations in ABL1 identified in primary NSCLC tumors and a lung cancer cell line increase downstream pathway activation compared to wild-type ABL1. Finally, we observe that the ABL1 cancer mutants display an increased cytosolic localization, which is associated with the oncogenic properties of the ABL1 kinase. In summary, our results suggest that NSCLC patients with ABL1 mutations could be stratified for treatment with imatinib in combination with other therapies.

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

Using large-scale genomics data to identify driver mutations in lung cancer: methods and challenges.

Lung cancer is the commonest cause of cancer death in the world and carries a poor prognosis for most patients. While precision targeting of mutated proteins has given some successes for never- and light-smoking patients, there are no proven targeted therapies for the majority of smokers with the disease. Despite sequencing hundreds of lung cancers, known driver mutations are lacking for a majority of tumors. Distinguishing driver mutations from inconsequential passenger mutations in a given lung tumor is extremely challenging due to the high mutational burden of smoking-related cancers. Here we discuss the methods employed to identify driver mutations from these large datasets. We examine different approaches based on bioinformatics, in silico structural modeling and biological dependency screens and discuss the limitations of these approaches.

Cancer-associated protein kinase C mutations reveal kinase's role as tumor suppressor.

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCß mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCß is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.

Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors.

RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2-18 months. Here we demonstrate that the mixed lineage kinases (MLK1-4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1-4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors.

Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer.

Approximately 70% of patients with non-small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ~50% of non-small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability.

Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region.

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force "barrier" on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRAB linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.