scholarly journals A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

2017 ◽  
Author(s):  
Thapakorn Jaroentomeechai ◽  
Xiaolu Zheng ◽  
Jasmine Hershewe ◽  
Jessica C. Stark ◽  
Michael C. Jewett ◽  
...  
Keyword(s):  
Protein Glycosylation ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Characterization Methods ◽  
In Vitro Characterization ◽  
Domains Of Life ◽  
Higher Eukaryotes ◽  
Single Subunit

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of post-translational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily-adaptable pipeline for virtually any ssOST of interest.

scholarly journals Bacterial flavin homeostasis: the role of an FAD pyrophosphatase/FMN transferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C311-C311
Author(s):  
Diana Tomchick ◽  
Ranjit Deka ◽  
Chad Brautigam ◽  
Wei Liu ◽  
Michael Norgard
Keyword(s):  
Pathogenic Bacteria ◽  
Treponema Pallidum ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
New Paradigm ◽  
Uptake Mechanism ◽  
Structural Investigations

Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter [1]. However, there is a paucity of information about flavin utilization in bacterial periplasms. We have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase/FMN transferase within the ApbE superfamily [2,3]. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). Treponemal Ftp (Ftp_Tp) is the first structurally and biochemically characterized metal-dependent FAD pyrophosphatase/FMN transferase in bacteria. We have shown in vitro and in vivo that Ftps from several types of pathogenic bacteria are capable of flavinylating proteins through covalent attachment of FMN via a phosphoester bond to threonine residues of an appropriate sequence signature. Progress on the structural characterization of a product of this post-translational modification will be presented. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.

scholarly journals SENP Proteases as Potential Targets for Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2059
Author(s):  
Paulina Tokarz ◽  
Katarzyna Woźniak
Keyword(s):  
Cancer Cells ◽  
Therapeutic Potential ◽  
Cysteine Proteases ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Small Molecular Weight ◽  
Aberrant Expression

SUMOylation is a reversible post-translational modification (PTM) involving a covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. SUMO-specific proteases (SENPs) are cysteine proteases with isopeptidase activity facilitating the de-conjugation of SUMO proteins and thus participating in maintaining the balance between the pools of SUMOylated and unSUMOylated proteins and in SUMO recycling. Several studies have reported that SENPs’ aberrant expression is associated with the development and progression of cancer. In this review, we will discuss the role of SENPs in the pathogenesis of cancer, focusing on DNA repair and the cell cycle—cellular pathways malfunctioning in most cancer cells. The plausible role of SENPs in carcinogenesis resulted in the design and development of their inhibitors, including synthetic protein-based, peptide-based, and small molecular weight inhibitors, as well as naturally occurring compounds. Computational methods including virtual screening have been implemented to identify a number of lead structures in recent years. Some inhibitors suppressed the proliferation of prostate cancer cells in vitro and in vivo, confirming that SENPs are suitable targets for anti-cancer treatment. Further advances in the development of SENP-oriented inhibitors are anticipated toward SENP isoform-specific molecules with therapeutic potential.

Differential S-acylation of Enveloped Viruses

2019 ◽  
Vol 26 (8) ◽  
pp. 588-600
Author(s):  
Larisa V. Kordyukova ◽  
Marina V. Serebryakova ◽  
Vladislav V. Khrustalev ◽  
Michael Veit
Keyword(s):  
Fatty Acid ◽  
Protein Trafficking ◽  
Virus Assembly ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Enveloped Virus ◽  
Infected Cells ◽  
Regulate Protein

Post-translational modifications often regulate protein functioning. Covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage (known as protein palmitoylation or S-acylation) affects protein trafficking, protein-protein and protein-membrane interactions. This post-translational modification is coupled to membrane fusion or virus assembly and may affect viral replication in vitro and thus also virus pathogenesis in vivo. In this review we outline modern methods to study S-acylation of viral proteins and to characterize palmitoylproteomes of virus infected cells. The palmitoylation site predictor CSS-palm is critically tested against the Class I enveloped virus proteins. We further focus on identifying the S-acylation sites directly within acyl-peptides and the specific fatty acid (e.g, palmitate, stearate) bound to them using MALDI-TOF MS-based approaches. The fatty acid heterogeneity/ selectivity issue attracts now more attention since the recently published 3D-structures of two DHHC-acyl-transferases gave a hint how this might be achieved.

scholarly journals A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rongsui Gao ◽  
Wenhui Wei ◽  
Bachar H Hassan ◽  
Jun Li ◽  
Jiaoyu Deng ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Common Mechanism ◽  
Acetyl Phosphate ◽  
Post Translational Modification ◽  
Positive Bacterium ◽  
Nad Metabolism ◽  
Domains Of Life

Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all domains of life, and its homeostasis must be regulated tightly. Here we report that a Nudix-related transcriptional factor, designated MsNrtR (MSMEG_3198), controls the de novo pathway of NAD+biosynthesis in M. smegmatis, a non-tuberculosis Mycobacterium. The integrated evidence in vitro and in vivo confirms that MsNrtR is an auto-repressor, which negatively controls the de novo NAD+biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, and can be disrupted by an ADP-ribose intermediate. Unexpectedly, we discover that the acetylation of MsNrtR at Lysine 134 participates in the homeostasis of intra-cellular NAD+ level in M. smegmatis. Furthermore, we demonstrate that NrtR acetylation proceeds via the non-enzymatic acetyl-phosphate (AcP) route rather than by the enzymatic Pat/CobB pathway. In addition, the acetylation also occurs on the paralogs of NrtR in the Gram-positive bacterium Streptococcus and the Gram-negative bacterium Vibrio, suggesting that these proteins have a common mechanism of post-translational modification in the context of NAD+ homeostasis. Together, these findings provide a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD+ metabolism.

scholarly journals Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

2018 ◽  
Author(s):  
Yin Yao Dong ◽  
Hua Wang ◽  
Ashley C.W. Pike ◽  
Stephen A. Cochrane ◽  
Sadra Hamedzadeh ◽  
...  
Keyword(s):  
Protein Glycosylation ◽  
Congenital Myasthenic Syndrome ◽  
List Type ◽  
Binding Modes ◽  
Post Translational Modification ◽  
Mechanism Of Catalysis ◽  
Myasthenic Syndrome ◽  
Essential Enzyme

SummaryProtein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.HighlightsStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysisDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activityStructures, kinetics and biosynthesis reveal role of lipid in tunicamycinLipid-altered, tunicamycin analogues give non-toxic antibiotics against TB

scholarly journals Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells

2017 ◽  
Vol 474 (14) ◽  
pp. 2489-2508 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Sew Yeu Peak-Chew ◽  
Clare Newell ◽  
Sheritta Miller-Aidoo ◽  
Sriyash Mangal ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Redox Regulation ◽  
Coenzyme A ◽  
Regulation Of Gene Expression ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Wide Range

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

scholarly journals State-of-the-art methods and devices for generation, exposure, and collection of aerosols from e-vapor products

2020 ◽  
Vol 4 ◽  
pp. 239784732097975
Author(s):  
Stéphanie Boué ◽  
Didier Goedertier ◽  
Julia Hoeng ◽  
Anita Iskandar ◽  
Arkadiusz K Kuczaj ◽  
...  
Keyword(s):  
Ad Hoc ◽  
State Of The Art ◽  
Physicochemical Characterization ◽  
Objective Evaluation ◽  
Vapor Generation ◽  
Characterization Methods ◽  
Clinical Exposure ◽  
Aerosol Generation

E-vapor products (EVP) have become popular alternatives for cigarette smokers who would otherwise continue to smoke. EVP research is challenging and complex, mostly because of the numerous and rapidly evolving technologies and designs as well as the multiplicity of e-liquid flavors and solvents available on the market. There is an urgent need to standardize all stages of EVP assessment, from the production of a reference product to e-vapor generation methods and from physicochemical characterization methods to nonclinical and clinical exposure studies. The objective of this review is to provide a detailed description of selected experimental setups and methods for EVP aerosol generation and collection and exposure systems for their in vitro and in vivo assessment. The focus is on the specificities of the product that constitute challenges and require development of ad hoc assessment frameworks, equipment, and methods. In so doing, this review aims to support further studies, objective evaluation, comparison, and verification of existing evidence, and, ultimately, formulation of standardized methods for testing EVPs.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

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