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

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.

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Extracellular endosulfatase Sulf-2 harbours a chondroitin/dermatan sulfate chain that modulates its enzyme activity

10.1101/2021.01.04.425218 ◽

2021 ◽

Author(s):

Rana El Masri ◽

Amal Seffouh ◽

Caroline Roelants ◽

Ilham Seffouh ◽

Evelyne Gout ◽

...

Keyword(s):

Enzyme Activity ◽

Dermatan Sulfate ◽

Post Translational Modification ◽

New Paradigm ◽

Extracellular Milieu ◽

Enzyme Substrate ◽

Enzyme Substrate Recognition ◽

Substrate Binding Domain

AbstractSulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes.

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SENP Proteases as Potential Targets for Cancer Therapy

Cancers ◽

10.3390/cancers13092059 ◽

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.

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A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

10.1101/155101 ◽

2017 ◽

Cited By ~ 1

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.

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Differential S-acylation of Enveloped Viruses

Protein and Peptide Letters ◽

10.2174/0929866526666190603082521 ◽

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.

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Synthesis and Characterization of Chemical Compounds Derived From Benzohydrazide and Evaluation of Their Antibacterial Activities

Avicenna Journal of Clinical Microbiology and Infection ◽

10.34172/ajcmi.2021.02 ◽

2021 ◽

Vol 8 (1) ◽

pp. 5-10

Author(s):

Maryam Alizadeh ◽

Ashraf Kariminik ◽

Ali Akbari

Keyword(s):

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Choline Chloride ◽

Antibacterial Activities ◽

Simple Method ◽

Major Health Problem ◽

Ft Ir

Background: The antimicrobial resistance of pathogenic bacteria has emerged as a major health problem in recent years. Extensive research has been conducted to find new antimicrobial agents. Objectives: The aim of this study was to examine the antibacterial activities of benzohydrazide derivatives. Methods: Manganese hydrogen sulfate choline chloride was applied in a simple method for synthesizing benzohydrazide derivatives. Antibacterial activities of the derivatives were assessed against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, diphtheroids, Salmonella enterica, Serratia marcescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The structure of the synthesized compounds was determined employing 1 H/13C NMR and Fourier-transform infrared (FT-IR) spectroscopy. The reactions were carried out in choline chloride dissolved in water at room temperature. Results: The results of this study showed that benzohydrazide derivatives had very desired antibacterial activities against the assessed bacteria. Conclusions: Further investigations are required to assess the safety and efficacy of benzohydrazide derivatives as antibacterial agents in vivo and in vitro.

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

Biochemical Journal ◽

10.1042/bcj20170129 ◽

2017 ◽

Vol 474 (14) ◽

pp. 2489-2508 ◽

Cited By ~ 37

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.

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In vivo and in vitro characterization of immediate release dosage forms containing n-acetylcysteine using the dynamic open flow-through test apparatus

10.26226/morressier.57d6b2b7d462b8028d88dd29 ◽

2016 ◽

Author(s):

Maximilian Sager

Keyword(s):

Immediate Release ◽

Dosage Forms ◽

Test Apparatus ◽

Open Flow ◽

In Vitro Characterization ◽

Flow Through

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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