Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Sugar transporters spatially organize microbiota colonization along the longitudinal root axis of Arabidopsis.

Plant roots are functionally heterogeneous in cellular architecture, transcriptome profile, metabolic state, and microbial immunity. We hypothesized that axial differentiation may also impact spatial colonization by root microbiota along the root axis. We developed two growth systems, ArtSoil and CD-Rhizotron, to grow and then dissect Arabidopsis thaliana roots into three segments. We demonstrate that distinct endospheric and rhizosphere bacterial communities colonize the segments, supporting the hypothesis of microbiota differentiation along the axis. Root metabolite profiling of each segment reveals differential metabolite enrichment and specificity. Bioinformatic analyses and GUS histochemistry indicate microbe-induced accumulation of SWEET2, 4, and 12 sugar uniporters. Profiling of root segments from sweet mutants shows altered spatial metabolic profiles and reorganization of endospheric root microbiota. This work reveals the interdependency between root metabolites and microbial colonization and the contribution of SWEETs to spatial diversity and stability of microbial ecosystem.

Effects of ScRgt1-Like DNA-binding transcription factor SpRgt1 (SPCC320.03) on Hexose transporters gene expression in Schizosaccharomyces pombe.

Glucose, which plays an essential role in carbon and energy metabolism in eukaryotes, is vital in directing various energy-consuming cellular processes. In S. cerevisiae, transcription factors involved in regulating hexose transporters and their mechanisms of action under different carbon sources were revealed in detail. However, there is limited information on these processes in S. pombe. In this study, the effect of SPCC320.03 (named SpRgt1), the ortholog of ScRgt1 whose molecular mechanism is known in detail in S. cerevisiae, on the transcriptional regulation of hexose transporters (ght1-8) dependent on different carbon sources was investigated. We measured the transcript levels of ght1-8 using the qPCR technique and performed relative evaluation in S. pombe strains (parental, rgt1 deleted mutant, rgt1 overexpressed, and vectoral rgt1 carrying mutant). We aimed to investigate the transcriptional changes caused by the protein product of the rgt1 (SPCC320.03) gene in terms of ght1-8 genes in strains that are grown in different carbon sources (2% glucose, 2% glycerol + 0.1% glucose, and 2% gluconate). Here, we show that SpRgt1 is involved in the regulation of the ght3, ght4, ght6, and ght7 genes but that the ght1, ght2, ght5, and ght8 gene expression vary depending on carbon sources, independently of SpRgt1.

Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and ß-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype.

Background: Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. Objective: We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. Methods: In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Results: Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-ß-induced EMT biomarker SNAIL and cell chemotaxis. Conclusion: Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.

Maltose-Derivatized Fluorescence Turn-On Imaging Probe for Bacteria Detection.

We report a maltose-derivatized fluorescence turn-on imaging probe, Mal-Cz, to detect E. coli and Staphylococci. The fluorescence turn-on is achieved through an intramolecular C-H insertion reaction of the perfluoroaryl azide-functionalized carbazole to give a fluorescent product. Confocal fluorescence microscopy confirmed the successful uptake of Mal-Cz by E. coli and Staphylococci upon photoactivation. The Mal-Cz probe could selectively detect E. coli and S. epidermidis in the presence of P. aeruginosa and M. smegmatis without interference from these bacteria. Both the photoactivation and bacteria detection can be accomplished using a hand-held UV lamp at 365 nm, with the limit of detection of 103 CFU/mL by the naked eye. Mal-Cz could also be used to detect E. coli and S. epidermidis spiked in milk by the naked eye under a hand-held UV lamp. The uptake of Mal-Cz requires metabolically active bacteria: the uptake was reduced in stationary phase bacteria and was diminished in bacteria that were killed by heating or treating with antibiotics or sodium azide. The uptake decreased with increasing concentration of added free maltose, indicating that Mal-Cz hijacked the maltose uptake pathways. In E. coli, the maltose transport systems, including maltoporin LamB, maltose binding protein MBP, and the maltose ATP binding cassette (ABC) transporter MalFGK2, are all critical for the transport of Mal-Cz. The uptake was diminished in the deletion mutants ΔLamB, ΔMalE, ΔMalF, and ΔMalK.

The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization.

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter's substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.

The role of salt bridge networks in the stability of the yeast hexose transporter 1.

BACKGROUND: The yeast S. cerevisiae preferably metabolizes glucose through aerobic glycolysis. Glucose transport is facilitated by multiple hexose transporters (Hxts), and their expression and activity are tightly regulated by multiple mechanisms. However, detailed structural and functional analyses of Hxts remain limited, largely due to the lack of crystal structure. METHODS: Homology modeling was used to build a 3D structural model for the yeast glucose transporter Hxt1 and investigate the effects of site directed mutations on Hxt1 stability and glucose transport activity. RESULTS: The conserved salt bridge-forming residues observed in the human Glut4 and the yeast glucose receptor Rgt2 were identified within and between the two 6-transmembrane spanning segments of Hxt1. Most of the RGT2 mutations that disrupt the salt bridge networks were known to cause constitutive signal generation, whereas the corresponding substitutions in HXT1 were shown to decrease Hxt1 stability. While substitutions of the two residues in the salt bridge 2 in Glut4-E329Q and E393D-were reported to abolish glucose transport, the equivalent substitutions in Hxt1 (D382Q and E454D) did not affect Hxt1 glucose transport activity. CONCLUSIONS: Substitutions of equivalent salt bridge-forming residues in Hxt1, Rgt2, and Glut4 are predicted to lock them in an inward-facing conformation but lead to different functional consequences. GENERAL SIGNIFICANCE: The salt bridge networks in yeast and human glucose transporters and yeast glucose receptors may play different roles in maintaining their structural and functional integrity.

The glycosylation defect in solute carrier SLC35A2/SLC35A3 double knockout cells is rescued by SLC35A2-SLC35A3 and SLC35A3-SLC35A2 hybrids.

SLC35A2 and SLC35A3 are homologous proteins with postulated nucleotide sugar transporting activities. Unlike SLC35A2, whose specificity for UDP-Gal is well-established, the UDP-GlcNAc transporting activity initially attributed to SLC35A3 has recently been put into question. In this study, we constructed two hybrid proteins (SLC35A2-SLC35A3 and SLC35A3-SLC35A2) and expressed them in a previously generated SLC35A2/SLC35A3 double knockout HEK293T cell line. Our idea was to force equivalent stoichiometry of the two proteins in the cells in order to reproduce the behavior of the SLC35A2/SLC35A3 complexes in the Golgi membrane. The hybrid proteins were able to fully restore glycosylation in the double knockout. In contrast, the expression of SLC35A3 alone in these cells improved galactosylation only to a limited extent. Our study shows that the proper glycosylation requires a balanced cooperation between SLC35A2 and SLC35A3.

Vacuolar sugar transporter EARLY RESPONSE TO DEHYDRATION6-LIKE4 affects fructose signaling and plant growth.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by 2 further observations. First, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and second, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

CLEC16A-An Emerging Master Regulator of Autoimmunity and Neurodegeneration.

CLEC16A is emerging as an important genetic risk factor for several autoimmune disorders and for Parkinson disease (PD), opening new avenues for translational research and therapeutic development. While the exact role of CLEC16A in health and disease is still being elucidated, the gene plays a critical role in the regulation of autophagy, mitophagy, endocytosis, intracellular trafficking, immune function, and in biological processes such as insulin secretion and others that are important to cellular homeostasis. As shown in both human and animal modeling studies, CLEC16A hypofunction predisposes to both autoinflammatory phenotype and neurodegeneration. While the two are clearly related, further functional studies are needed to fully understand the mechanisms involved for optimized therapeutic interventions. Based on recent data, mitophagy-inducing drugs may be warranted, and such therapy should be tested in clinical trials as these drugs would tackle the underlying pathogenic mechanism (s) and could treat or prevent symptoms of autoimmunity and neurodegeneration in individuals with CLEC16A risk variants. Accordingly, interventions directed at reversing the dysregulated mitophagy and the consequences of loss of function of CLEC16A without activating other detrimental cellular pathways could present an effective therapy. This review presents the emerging role of CLEC16A in health and disease and provides an update on the disease processes that are attributed to variants located in the CLEC16A gene, which are responsible for autoimmune disorders and neurodegeneration with emphasis on how this information is being translated into practical and effective applications in the clinic.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.

Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network.

Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+ -D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.

Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and CD spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

An in silico quest for next-generation antimalarial drugs by targeting Plasmodium falciparum hexose transporter protein: a multi-pronged approach.

The emergence of artemisinin resistance by malaria parasites is a major challenge in the fight against malaria, thus posing serious threat to the public health across the world. To tackle this, antimalarial drugs with unconventional mechanisms are therefore urgently needed. It has been reported that selective starvation of Plasmodium falciparum by blocking the function of hexose transporter 1 (PfHT1) protein, the only known transporter for glucose uptake in P. falciparum, could provide an alternative approach to fight the drug resistant malaria parasites. In this study, three high affinity molecules (BBB_25784317, BBB_26580136 and BBB_26580144) that have shown the best docked conformation and least binding energy with PfHT1 were shortlisted. The docking energy of BBB_25784317, BBB_26580136 and BBB_26580144 with PfHT1 were -12.5, -12.1 and -12.0 kcal/mol, respectively. In the follow up simulation studies, the protein 3D structure maintains considerable stability in the presence of the compounds. It was also observed that the compounds produced a number of hydrophilic and hydrophobic interactions with the protein allosteric site residues. This demonstrates strong intermolecular interaction guided by close distance hydrogen bonds of compounds with Ser45, Asn48, Thr49, Asn52, Ser317, Asn318, Ile330 and Ser334. Revalidation of compounds binding affinity was conducted by more appropriate simulation based binding free energy techniques (MM-GB/PBSA and WaterSwap). Additionally, entropy assay was performed that further strengthen the predictions. In silico pharmacokinetics confirmed that the compounds would be suitable candidates for oral delivery due to their high gastrointestinal absorption and less toxic reaction. Overall, the predicted compounds are promising and could be further sought as antimalarial leads and subjected to thorough experimental investigations.Communicated by Ramaswamy H. Sarma.

CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment.

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.

Synthetic Monosaccharide Channels: Size-Selective Transmembrane Transport of Glucose and Fructose Mediated by Porphyrin Boxes.

Here we report synthetic monosaccharide channels built with shape-persistent organic cages, porphyrin boxes (PBs), that allow facile transmembrane transport of glucose and fructose through their windows. PBs show a much higher transport rate for glucose and fructose over disaccharides such as sucrose, as evidenced by intravesicular enzyme assays and molecular dynamics simulations. The transport rate can be modulated by changing the length of the alkyl chains decorating the cage windows. Insertion of a linear pillar ligand into the cavity of PBs blocks the monosaccharide transport. In vitro cell experiment shows that PBs transport glucose across the living-cell membrane and enhance cell viability when the natural glucose transporter GLUT1 is blocked. Time-dependent live-cell imaging and MTT assays confirm the cyto-compatibility of PBs. The monosaccharide-selective transport ability of PBs is reminiscent of natural glucose transporters (GLUTs), which are crucial for numerous biological functions.

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model.

Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or glucose obtainable from cellulose. P. putida EM42, a genome-reduced variant of strain KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose and glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the co-utilization phenotype remained to be understood and the Δgcd strain had a significant growth defect. In this study, we investigated the basis of the simultaneous uptake of the two sugars and accelerated the growth of P. putida EM42 Δgcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion lifted the inhibition of the exogenous ß-glucosidase BglC from Thermobifida fusca exerted by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore rapid growth on glucose. The reduced growth rate of the Δgcd mutant was partially compensated by the implantation of heterologous glucose and cellobiose transporters (Glf from Zymomonas mobilis and LacY from Escherichia coli, respectively). Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The pyruvate overproduction phenotype was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions.

Nucleotide sugar transporter SLC35A2 is involved in promoting hepatocellular carcinoma metastasis by regulating cellular glycosylation.

PURPOSE: Recently, aberrant glycosylation has been recognized to be relate to malignant behaviors of cancer and outcomes of patients with various cancers. SLC35A2 plays an indispensable role on glycosylation as a nucleotide sugar transporter. However, effects of SLC35A2 on malignant behaviors of cancer cells and alteration of cancer cells surface glycosylation profiles are still not fully understood, particularly in hepatocellular carcinoma (HCC). Hence, from a glycosylation perspective, we investigated the effects of SLC35A2 on metastatic behaviors of HCC cells. METHODS: SLC35A2 expression in clinical samples and HCC cells was examined by immunohistochemical staining or Western blot/quantitative PCR and was regulated by RNA interference or vectors-mediated transfection. Effects of SLC35A2 expression alteration on metastatic behaviors and membrane glycan profile of HCC cells were observed by using respectively invasion, migration, cell adhesion assay, in vivo lung metastatic nude mouse model and lectins microarray. Co-location among proteins in HCC cells was observed by fluorescence microscope and detected by an in vitro co-immunoprecipitation assay. RESULTS: SLC35A2 was upregulated in HCC tissues, and is associated with poor prognosis of HCC patients. SLC35A2 expression alteration significantly affected the invasion, adhesion, metastasis and membrane glycan profile and led to the dysregulated expressions or glycosylation of cell adhesion-related molecules in HCC cells. Mechanistically, the maintenance of SLC35A2 activity is critical for the recruitment of the key galactosyltransferase B4GalT1, which is responsible for complex glycoconjugate and lactose biosynthesis, to Golgi apparatus in HCC cells. CONCLUSION: SLC35A2 plays important roles in promoting HCC metastasis by regulating cellular glycosylation modification and inducing the cell adhesive ability of HCC cells.

An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. IDPRs comprise nearly half of the human proteome, yet their mechanistic roles in human disease are poorly understood. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases CLEC16A ubiquitination and degradation, thus impairing assembly of the mitophagy regulatory machinery. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we elucidate how an IDPR in CLEC16A regulates mitophagy and implicate pathogenic human gene variants that disrupt IDPRs as novel contributors to diabetes and other CLEC16A-associated diseases.Abbreviations : CAS: carbon-detect amino-acid specific; IDPR: intrinsically disordered protein region; MEFs: mouse embryonic fibroblasts; NMR: nuclear magnetic resonance.