Posttranslational Modification Enzymes Research Articles

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Tailoring Multipurpose Biocatalysts via Protein Engineering Approaches: A Review

The current industrial revolution signifies the high-value of protein engineering. The development of multipurpose biocatalysts is significantly expanding as a result of increased access and enzyme tailoring ability to satisfy the ever-increasing industrial demands. Enzyme-catalyzed processes offers multi-benefits at a time, e.g., low catalyst loading, high specificity, selectivity, mild processing for a complex and chemically unstable compounds, capability to reduce or eliminate reaction by-products, overall reusability and cost-effective ratio via immobilization, and potential to carry out conventional multi-stage processes via one-pot reaction. In this review, we critically elaborated recent achievements in applying new and/or state-of-the-art sophisticated protein engineering approaches to tailor the catalytic properties of enzymes or design enzymes with new and improved activities to catalyze desired biochemical transformations by orders of magnitude. We focused on different protein engineering approaches such as substrate engineering, medium engineering, and post-translational enzyme modification, structure-assisted protein tailoring, advanced computational modeling, and the exploration of inimitable synthetic scaffolds to develop multipurpose biocatalyst and improve the performance of multi-enzyme systems. In short, this study demonstrates an array of molecular biology insights and computational designs speeding up the tailored design of new and industrial biocatalysts. Continuous key developments in this direction together with protein engineering in unique ways might offer the ever-increasing opportunities for impending biocatalysis research for industrial bioprocesses.

Unraveling the activity of glycosyltransferases and other PTM enzymes with bioluminescent biochemical and cell based assays

Post translational modifications (PTMs) are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, glycosylation, and ubiquitination, and influence many aspects of normal cell biology and pathogenesis. Examples of post translational modification enzymes include but are not limited to, kinases/phosphatases, methyltransferases/demethylases, and glycosyltransferases/glucanases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzyme activities can be dysregulated, and the disruption of the intracellular networks they govern leads to an array of diseases including cancer and inflammation. Consequently, PTM enzymes have become important targets for drug discovery creating a need for development of activity detection assays. Current assays to analyze cellular or biochemical PTM enzyme activities can be tedious, non‐homogeneous, and not easily adaptable to HTS. They can also suffer from generation of false hits due to compound interference or require special instruments for detection. To overcome these shortcomings, we developed an array of biochemical and cellular bioluminescent assays to detect these enzyme activities. This presentation will showcase how cellular mechanisms driven by PTM in general and phosphorylation and glcnacylation in particular can be addressed by analyzing these enzyme's cellular activity using the NanoLuc Binary Technology (NanoBiT), a two‐subunit complementation system based on NanoLuc luciferase. We will also discuss how the bioluminescent nucleotide assays that detect UDP, GDP, UMP and CMP as means to measure glycosyltransferase activities can streamline studies on GT specificity of transfer of different sugars to different acceptors. We will show their use in screening for specific inhibitors as well as in studies of their modes of action. Development of these bioluminescent detection assays will enable the investigation of key signaling pathways and the study of many PTM enzymes in general and GTs in particular, which may have significant impact on diverse areas of glycobiology research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deubiquitylase USP7 regulates human terminal erythroid differentiation by stabilizing GATA1.

Ubiquitination is an enzymatic post-translational modification that affects protein fate. The ubiquitin-proteasome system (UPS) was first discovered in reticulocytes where it plays important roles in reticulocyte maturation. Recent studies have revealed that ubiquitination is a dynamic and reversible process and that deubiquitylases are capable of removing ubiquitin from their protein substrates. Given the fact that the UPS is highly active in reticulocytes, it is speculated that deubiquitylases may play important roles in erythropoiesis. Yet, the role of deubiquitylases in erythropoiesis remains largely unexplored. In the present study, we found that the expression of deubiquitylase USP7 is significantly increased during human terminal erythroid differentiation. We further showed that interfering with USP7 function, either by short hairpin RNA-mediated knockdown or USP7-specific inhibitors, impaired human terminal erythroid differentiation due to decreased GATA1 level and that restoration of GATA1 levels rescued the differentiation defect. Mechanistically, USP7 deficiency led to a decreased GATA1 protein level that could be reversed by proteasome inhibitors. Furthermore, USP7 interacts directly with GATA1 and catalyzes the removal of K48-linked poly ubiquitylation chains conjugated onto GATA1, thereby stabilizing GATA1 protein. Collectively, our findings have identified an important role of a deubiquitylase in human terminal erythroid differentiation by stabilizing GATA1, the master regulator of erythropoiesis.

Lysyl hydroxylase 3 increases collagen deposition and promotes pulmonary fibrosis by activating TGFβ1/Smad3 and Wnt/β-catenin pathways

IntroductionLysyl hydroxylase 3 (LH3) is a collagen post-translational modifying enzyme; it is abnormally activated during the formation of collagen cross-links. iCRT3 is an inhibitor of both Wnt and β-catenin responsive transcription. We hypothesized that LH3 is regulated by TGFβ1/Smad3 signaling and Wnt/β-catenin signaling pathways. Some evidence suggested that there is complicated cross-talk between the two signal pathways in the genesis of pulmonary fibrosis.Material and methodsThe normal culturing human lung cancer cell line A549 was derived from pulmonary epithelial cells. Transforming growth factor-β1 (TGF-β1) was induced A549 cells of pulmonary fibrosis. MTT assays detected cell growth stimulation by TGF-β1; collagen pyridine-crosslinking contents were detected by ELISA kits. Immunofluorescence were used to evaluate expression of key molecules in PLOD3 (LH3), Wnt/β-catenin and TGFβ1/Smad3 pathways.ResultsOur findings suggested that iCRT3 could decrease LH3 protein expression (p < 0.01), Wnt1, β-catenin and p-Smad3 protein expression (p < 0.05). Knock-down PLOD3 could decrease LH3, collagen I gene and protein expression (p < 0.05). These effects were associated with decreasing collagen pyridine-crosslinking production (p < 0.05). However, ovexpression PLOD3 could increase LH3, collagen I gene and protein expression (p < 0.05). The result showed that LH3 plays an important role in collagen post-translational modifications, and it is regulated by Wnt/β-catenin and TGFβ1/Smad3 pathways.ConclusionsThis study suggests that PLOD3 (LH3) represents a target to prevent pulmonary fibrosis.

Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides

We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short <20 amino acid peptides without limitations rising from the leucine-zipper coiled-coil structure. Introduced methodology enables wash-free PTM detection in a 384-well plate format, using low nanomolar enzyme concentrations. Potentially, the peptide-break technique using charged peptides may be applicable for natural peptide sequences directly obtained from the target protein.

Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer.

: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

Advanced glycation endproducts and polysialylation affect the turnover of the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation endproducts (RAGE).

The balance between protein synthesis and degradation regulates the amount of expressed proteins. This protein turnover is usually quantified as the protein half-life time. Several studies suggest that protein degradation decreases with age and leads to increased deposits of damaged and non-functional proteins. Glycation is an age-dependent, non-enzymatic process leading to posttranslational modifications, so-called advanced glycation endproducts (AGE), which usually damage proteins and lead to protein aggregation. AGE are formed by the Maillard reaction, where carbonyls of carbohydrates or metabolites react with amino groups of proteins. In this study, we quantified the half-life time of two important receptors of the immunoglobulin superfamily, the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation end products (RAGE) before and after glycation. We found, that in two rat PC12 cell lines glycation leads to increased turnover, meaning that glycated, AGE-modified proteins are degraded faster than non-glycated proteins. NCAM is the most prominent carrier of a unique enzymatic posttranslational modification, the polysialylation. Using two PC12 cell lines (a non-polysialylated and a polysialylated one), we could additionally demonstrate, that polysialylation of NCAM has an impact on its turnover and that it significantly increases its half-life time.

Abstract 5899: Genome-wide CRISPR-Cas9 screening identifies genetic vulnerabilities and potential therapeutic targets in castration resistant prostate cancer

Abstract Background: Treatment options for castration-resistant prostate cancer (CRPC) are limited and only a few agents (e.g. enzalutamide, docetaxel) are routinely used in the clinic. Unfortunately, CRPC tumors will invariable develop resistance to these agents and only a subset of patients will respond. Thus, novel predictive biomarkers are urgently needed to ensure that an expensive and potentially harmful agent is given only to patients who will benefit from it. Furthermore, to enable new treatment strategies, a better understanding of drug resistance mechanisms is required. Methods: To identify novel drug resistance genes and mechanisms of therapy resistance, we performed genome-wide CRISPR-Cas9 knockout screens in LNCaP (hormone naïve) and in the isogenic C4 (castration resistant) PC cell line, respectively. Using 77,441 unique sgRNAs (Brunello library), a total of 19,114 protein-coding genes were tested for their potential functional role in enzalutamide or docetaxel resistance. Both IC50 and IC90 values were used for selection. MAGeCK was used to identify enriched and depleted sgRNAs (treatment vs. DMSO). Results: Among the highest ranked hits for the C4 dropout screens, several potential genetic vulnerabilities and mechanisms of resistance were identified, as knockout of specific genes sensitized C4 cells to enzalutamide. None of these hits were observed in LNCaP, which suggest CRPC-specific resistance mechanisms. For the positive screens, increased enzalutamide resistance was observed after knockout of genes encoding e.g. phosphokinases and specific solute carriers (SLCs) for C4 and LNCaP, respectively. Lastly, our results suggest that knockout of specific zinc finger nucleases (ZFNs), posttranslational modification enzymes or microtubule components may modulate docetaxel resistance in C4 cells. Conclusion: Drug resistance is a major clinical problem. Here, we identified genetic vulnerabilities that may be translated into predictive biomarkers, combination therapies and/or novel drug development strategies for CRPC. Citation Format: Jakob Haldrup, Linnéa Schmidt, Jakob S. Pedersen, Karina D. Sørensen. Genome-wide CRISPR-Cas9 screening identifies genetic vulnerabilities and potential therapeutic targets in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5899.

Genomic comparisons of Rhizobium species using in silico AFLP-PCR, endonuclease restriction, and AMPylating enzymes

The whole-genome sequences of nine Rhizobium species were evaluated using different in silico molecular techniques such as AFLP-PCR, restriction digest, and AMPylating enzymes. The entire genome sequences were aligned with progressiveMauve and visualized by reconstructing phylogenetic tree using NTSYS pc 2.11X. The “insilico.ehu.es” was used to carry out in silico AFLP-PCR and in silico restriction digest of the selected genomes. Post-translational modification (PTM) and AMPylating enzyme diversity between the proteome of Rhizobium species were determined by novPTMenzy. Slight variations were observed in the phylogeny based on AFLP-PCR and PFGE and the tree based on whole genome. Results clearly demonstrated the presence of PTMs, i.e., AMPylation with the GS-ATasE (GlnE), Hydroxylation, Sulfation with their domain, and Deamidation with their specific domains (AMPylating enzymes) GS-ATasE (GlnE), Fic, and Doc (Phosphorylation); Asparagine_hydroxylase and Collagen_prolyl_lysyl_hydroxylase; Sulfotransferase; and CNF (Cytotoxic Necrotizing Factors), respectively. The results pertaining to PTMs are discussed with regard to functional diversities reported in these species. The phylogenetic tree based on AFLP-PCR was slightly different from restriction endonuclease- and PFGE-based trees. Different PTMs were observed in the Rhizobium species, and the most prevailing type of PTM was AMPylation with the domain GS-ATasE (GlnE). Another type of PTM was also observed, i.e., Hydroxylation and Sulfation, with the domains Asparagine_hydroxylase and Collagen_prolyl_lysyl_hydroxylase and Sulfotransferase, respectively. The deamidation type of PTM was present only in Rhizobium sp. NGR234. How to cite: Qureshi MA, Pervez MT, Babar ME, et al. Genomic comparisons of Rhizobium species using in silico AFLP-PCR, endonuclease restrictions and ampylating enzymes. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.05.006.

Genetically Encoded Protein Phosphorylation in Mammalian Cells

SummaryProtein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRSv1.0/tRNAv1.0CUA pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii [Mj]tRNAGCACys pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1α) variant, EF-1α-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell’s phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells.

Purification and Characterization of α-Mannosidase from Moss Hyophilla nymaniana (Fleish.) Menzel

α-Mannosidase EC: (3.2.1.24) is the major post-translational modifications enzyme which catalyzes the cleavage of the alpha form of mannose. Lysosomal α-mannosidase assists in the processing of complex sugars from glycoproteins. Defective or deficient α-mannosidase activity causes α-mannosidosis and leads to deterioration of the central nervous system in children. In the present study, plant α-mannosidase was purified 39-fold in a stepwise manner by ammonium sulfate precipitation, dialysis, gel filtration chromatography and DEAE ion exchange chromatography. The purified mannosidase was a heterodimer consisting of approximately 72 and 50 kDa molecular mass subunits under reducing conditions. Optimum pH and temperature reported for mannosidase activity were 4.5 and 40 °C respectively. Among the various metal ions tested for the assessment of the enzyme activity, 2 mM Zn2+ strongly activated the enzyme while Hg2+and Ag2+ caused enzyme inhibition at the same concentration. The Km and Vmax values calculated from Lineweaver plot against the substrate p-nitrophenyl α-mannopyranoside were 5.4 mM and 8.4 U/ml, respectively. To the author’s knowledge, the data presented here is the first report on purification and characterization of α-mannosidase from the moss H. nymaniana.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus.

RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.

Protein Palmitoylation and Its Role in Bacterial and Viral Infections.

S-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. S-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. In this review, we focus on S-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. We discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. We highlight recent data on protein S-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. The role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed.

Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency.

Collagen is one of the most important components of the extracellular matrix that is involved in the strength of tissues, cell adhesion and cell proliferation. Mutations in several collagen and post-translational modification enzyme genes cause Ehlers-Danlos syndrome (EDS) characterized by joint and skin hyperextensibility as well as fragility of various organs. Carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase-1 (CHST14/D4ST1) is a critical enzyme for biosynthesis of dermatan sulfate, a side chain of various proteoglycans including biglycan that regulates collagen fibrils through their interaction. Mutations in CHST14 were found to cause a new form of EDS, named musculocontractural type EDS (mcEDS-CHST14). Large subcutaneous hematomas are one of the most serious complications accompanied by decreased quality of life and potential lethality. In this study, Chst14 gene-deleted mice were expected to be an animal model of the vascular abnormalities of mcEDS-CHST14. However, only limited numbers of adult mice were generated because of perinatal lethality in most Chst14 gene-deleted homozygote (Chst14-/-) mice. Therefore, we investigated the placentas of these fetuses. The placentas of Chst14-/- fetuses showed a reduced weight, alterations in the vascular structure, and ischemic and/or necrotic-like changes. Electron microscopy demonstrated an abnormal structure of the basement membrane of capillaries in the placental villus. These findings suggest that Chst14 is essential for placental vascular development and perinatal survival of fetuses. Furthermore, placentas of Chst14-/- fetuses could be a useful model for vascular manifestations in mcEDS-CHST14, such as the large subcutaneous hematomas.

Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes.

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone–receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation.

Global substrate specificity profiling of post-translational modifying enzymes.

Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile.

Post-translational regulation of metabolism in fumarate hydratase deficient cancer cells

Deregulated signal transduction and energy metabolism are hallmarks of cancer and both play a fundamental role in tumorigenesis. While it is increasingly recognised that signalling and metabolism are highly interconnected, the underpinning mechanisms of their co-regulation are still largely unknown. Here we designed and acquired proteomics, phosphoproteomics, and metabolomics experiments in fumarate hydratase (FH) deficient cells and developed a computational modelling approach to identify putative regulatory phosphorylation-sites of metabolic enzymes. We identified previously reported functionally relevant phosphosites and potentially novel regulatory residues in enzymes of the central carbon metabolism. In particular, we showed that pyruvate dehydrogenase (PDHA1) enzymatic activity is inhibited by increased phosphorylation in FH-deficient cells, restricting carbon entry from glucose to the tricarboxylic acid cycle. Moreover, we confirmed PDHA1 phosphorylation in human FH-deficient tumours. Our work provides a novel approach to investigate how post-translational modifications of enzymes regulate metabolism and could have important implications for understanding the metabolic transformation of FH-deficient cancers with potential clinical applications.