Machine Learning-Supported Enzyme Engineering toward Improved CO2-Fixation of Glycolyl-CoA Carboxylase.

Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO2 fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO2 fixation. To further improve upon GCC M5, we developed a machine learning-supported workflow that reduces screening efforts for identifying improved enzymes. Using this workflow, we present two novel GCC variants with 2-fold increased carboxylation rate and 60% reduced energy demand, respectively, which are able to address kinetic and thermodynamic limitations of the TaCo pathway. Our work highlights the potential of combining machine learning and directed evolution strategies to reduce screening efforts in enzyme engineering.

C/EBP-Induced Transdifferentiation Reveals Granulocyte-Macrophage Precursor-like Plasticity of B Cells.

The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBPα, ß, δ, and ε) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBPα and ß activation. High transgene expression of C/EBPß or C/EBPε, but not of C/EBPα or C/EBPδ, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBPß-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells.

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.

DAPK-HSF1 interaction as a positive-feedback mechanism stimulating TNF-induced apoptosis in colorectal cancer cells.

Death-associated protein kinase (DAPK) is a serine-threonine kinase with tumor suppressor function. Previously, we demonstrated that tumor necrosis factor (TNF) induced DAPK-mediated apoptosis in colorectal cancer. However, the protein-protein interaction network associated with TNF-DAPK signaling still remains unclear. We identified HSF1 as a new DAPK phosphorylation target in response to low concentrations of TNF and verified a physical interaction between DAPK and HSF1 both in vitro and in vivo. We show that HSF1 binds to the DAPK promoter. Transient overexpression of HSF1 protein led to an increase in DAPK mRNA level and consequently to an increase in the amount of apoptosis. By contrast, treatment with a DAPK-specific inhibitor as well as DAPK knockdown abolished the phosphorylation of HSF1 at Ser230 (pHSF1(Ser230)). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1(Ser230) protein expression in human colorectal carcinoma tissues. Taken together, our data define a novel link between DAPK and HSF1 and highlight a positive-feedback loop in DAPK regulation under mild inflammatory stress conditions in colorectal tumors. For the first time, we show that under TNF the pro-survival HSF1 protein can be redirected to a pro-apoptotic program.

DAPK and cytoskeleton-associated functions.

Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser(19), thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser(188) in a Ca(2+) dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.

Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis.

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilin(Ser3)). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilin(Ser3) was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilin(Ser3) mutants we suggest that p-cofilin(Ser3) is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.

Death-associated protein kinase controls STAT3 activity in intestinal epithelial cells.

The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC.

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions.

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn't rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.

Apoptosis signalling activated by TNF in the lower gastrointestinal tract--review.

The tumor necrosis factor (TNF) gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving epigenetic mechanisms or miRNAs. A better knowledge of the molecular mechanisms that control TNF gene regulation and TNF signalling will provide deeper understanding of the initiation and development of apoptotic and inflammatory processes triggered by TNF cytokine in the gut. The described efforts to embed TNF in clinical treatment regiments reflect its attractive effectiveness in killing tumor cells. Whether the described strategies will achieve the success of incorporating TNF in lower gastrointestinal tract therapy for inflammatory diseases and cancer remains to be determined.

RETRACTED: Identification of phosphorylated p38 as a novel DAPK-interacting partner during TNFalpha-induced apoptosis in colorectal tumor cells.

Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signaling on cytokine exposure. The role of DAPK in macrophage-associated tumor cell death is currently unknown. Recently, we suggested a new function for DAPK in the induction of apoptosis during the interaction between colorectal tumor cells and tumor-associated macrophages. Using a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and human HCT116 colorectal tumor cells, we replicated DAPK-associated tumor cell death; this model likely reflects the in vivo tumor setting. In this study, we show that tumor necrosis factor-alpha exposure under conditions of macrophage activation induced DAPK-dependent apoptosis in the colorectal tumor cell line HCT116. Simultaneously, early phosphorylation of p38 mitogen-activated protein kinase (phospho-p38) was observed. We identified the phospho-p38 mitogen-activated protein kinase as a novel interacting protein of DAPK in tumor necrosis factor-alpha-induced apoptosis. The general relevance of this interaction was verified in two colorectal cell lines without functional p53 (ie, HCT116 p53(-/-) and HT29 mutant) and in human colon cancer and ulcerative colitis tissues. Supernatants of freshly isolated human macrophages were also able to induce DAPK and phospho-p38. Our findings highlight the mechanisms that underlie DAPK regulation in tumor cell death evoked by immune cells.