Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

High yield secretion of human erythropoietin from tobacco cells for ex vivo differentiation of hematopoietic stem cells towards red blood cells.

Human erythropoietin (EPO) is a key cytokine in erythropoiesis by regulating differentiation of erythroid progenitor cells into red blood cells (RBCs). Plant cell cultures are considered as promising alternative bioproduction platforms for EPO. To overcome the bottlenecks of low protein productivity and secretion, EPO was expressed in tobacco BY-2 cells with a designer peptide tag, termed (SP)20 that consists of 20 tandem repeats of a "Ser-Pro" motif. This de novo designed tag directed extensive O-glycosylation on each Pro residue in plant cells and acted as a molecular carrier to promote the extracellular secretion of EPO. To facilitate the establishment of stable and high-expression BY-2 cell lines, EPO molecules were co-expressed with a reporter protein GFP, which could be used as a visual marker to monitor the protein expression during the subculture. The engineered (SP)20 glycomodule substantially increased the secreted yields of EPO up to 4.31 µg/mL. The (SP)20-tagged EPOs exhibited the expected activity in promoting the proliferation of TF-1 cells, though their EC50 was 12-fold higher than that of EPO standard. The (SP)20-tagged EPOs could also stimulate the ex vivo expansion and differentiation of hematopoietic stem cell (CD34+ cells) towards RBCs.

Engineering hydroxyproline-O-glycosylated biopolymers to reconstruct the plant cell wall for improved biomass processability.

Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)-O-glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp-O-glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of "Ser-Hyp-Hyp-Hyp-Hyp" motif or (SP4)18 was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) from Acidothermus cellulolyticus. The engineered (SP4)18 module was extensively Hyp-O-glycosylated with arabino-oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5-11-fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)18 module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)18 -tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5-fold without having adverse impact on the plant growth.

In Vitro Biocompatibility of Decellularized Cultured Plant Cell-Derived Matrices.

There has been a recent increase in exploring the use of decellularized plant tissue as a novel "green" material for biomedical applications. As part of this effort, we have developed a technique to decellularize cultured plant cells (tobacco BY-2 cells and rice cells) and tissue (tobacco hairy roots) that uses deoxyribonuclease I (DNase I)). As a proof of concept, all cultured plant cells and tissue were transformed to express recombinant enhanced green fluorescent protein (EGFP) to show that the proteins of interest could be retained within the matrices. Decellularization of lyophilized tobacco BY-2 cells with DNase for 30 min depleted the DNA content from 1503 ± 459 to 31 ± 5 ng/sample. The decellularization procedure resulted in approximately 36% total protein retention (154 ± 60 vs 424 ± 70 µg/sample) and 33% EGFP retention. Similar results for DNA removal and protein retention were observed with the rice cells and tobacco hairy root matrices. When exposed to decellularized BY-2 cell-derived matrices, monolayer cultures of human foreskin fibroblasts (hFFs) maintained or increased metabolic activity, which is an indicator of cell viability. Furthermore, hFFs were able to attach, spread, and proliferate when cultured with the decellularized BY-2 cell-derived matrices in an aggregate model. Overall, these studies demonstrate that cultured plant cells and tissue can be effectively decellularized with DNase I with substantial protein retention. The resulting material has a positive impact on hFF metabolic activity and could be employed to create a three-dimensional environment for cell growth. These results thus show the promise of using naturally derived cellulose matrices from cultured plant cells and tissues for biomedical applications.

Production of thermostable endo-1,5-α-L-arabinanase in Pichia pastoris for enzymatically releasing functional oligosaccharides from sugar beet pulp.

Sugar beet pulp is an agricultural processing residue that is a rich source of the cell wall polysaccharide arabinan. Functional oligosaccharides, specifically feruloylated arabino-oligosaccharides (FAOs), can be isolated from sugar beet pulp through selective action by endo-arabinanase (glycoside hydrolase family 43). This study aimed to develop yeast (Pichia pastoris) as an efficient, eukaryotic platform to produce a thermophilic endo-1,5-α-L-arabinanase (TS-ABN) for extracting FAOs from sugar beet pulp. Recombinant TS-ABN was secreted into yeast culture medium at a yield of ~ 80 mg/L, and the protein exhibited specific enzyme activity, pH and temperature optimum, and thermostability comparable to those of the native enzyme. Treatment of sugar beet pulp with Pichia-secreted TS-ABN released FAOs recovered by hydrophobic chromatography at 1.52% (w/w). The isolated FAOs averaged seven arabinose residues per ferulic acid, and treatment of T84 human colon epithelial cells significantly increased expression of two key tight junction-related proteins-zonula occludens-1 and occludin-in a dose-dependent manner. This research establishes a biochemical platform for utilizing sugar beet pulp to produce value-added bioproducts with potential nutraceutical applications.

Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.

Engineering 'designer' glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline-O-glycosylation process in plants.

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)-O-glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp-O-glycosylation process of plant cell wall Hyp-rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of 'Ser-Hyp-Hyp-Hyp-Hyp' motif or (SP4)18 and an arabinogalactan protein module consisting of 32 tandem repeats of 'Ser-Hyp' motif or (SP)32 . Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56-fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)18 and (SP)32 modules underwent Hyp-O-glycosylation with arabino-oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non-Hyp-O-glycosylated (SP4)18 -EGFP and (SP)32 -EGFP intermediates were consistently accumulated within the root tissues, indicating a rate-limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp-O-glycosylation and extracellular secretion of extensin-styled (SP4)18 module and AGP-styled (SP)32 module is proposed.