scholarly journals Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation

2012 ◽  
Vol 449 (2) ◽  
pp. 415-425 ◽  
Author(s):  
Mareike G. Posner ◽  
Abhishek Upadhyay ◽  
Susan J. Crennell ◽  
Andrew J. A. Watson ◽  
Steve Dorus ◽  
...  
Keyword(s):  
Conformational Change ◽  
Lipoic Acid ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Thermophilic Archaeon ◽  
C Complex ◽  
Biochemical Analyses ◽  
Enzyme Complexes ◽  
Lipoyl Domain

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N–LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N–LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N–LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN–LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N–LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N–LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.

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 Histone deacetylases (HDACs): characterization of the classical HDAC family

2003 ◽  
Vol 370 (3) ◽  
pp. 737-749 ◽  
Author(s):  
Annemieke J.M. de RUIJTER ◽  
Albert H. van GENNIP ◽  
Huib N. CARON ◽  
Stephan KEMP ◽  
André B.P. van KUILENBURG
Keyword(s):  
Gene Expression ◽  
Histone Deacetylases ◽  
Regulation Of Gene Expression ◽  
Hdac Inhibitors ◽  
Building Blocks ◽  
Post Translational Modification ◽  
Comprehensive Overview ◽  
Almost All ◽  
Enzyme Complexes

Transcriptional regulation in eukaryotes occurs within a chromatin setting, and is strongly influenced by the post-translational modification of histones, the building blocks of chromatin, such as methylation, phosphorylation and acetylation. Acetylation is probably the best understood of these modifications: hyperacetylation leads to an increase in the expression of particular genes, and hypoacetylation has the opposite effect. Many studies have identified several large, multisubunit enzyme complexes that are responsible for the targeted deacetylation of histones. The aim of this review is to give a comprehensive overview of the structure, function and tissue distribution of members of the classical histone deacetylase (HDAC) family, in order to gain insight into the regulation of gene expression through HDAC activity. SAGE (serial analysis of gene expression) data show that HDACs are generally expressed in almost all tissues investigated. Surprisingly, no major differences were observed between the expression pattern in normal and malignant tissues. However, significant variation in HDAC expression was observed within tissue types. HDAC inhibitors have been shown to induce specific changes in gene expression and to influence a variety of other processes, including growth arrest, differentiation, cytotoxicity and induction of apoptosis. This challenging field has generated many fascinating results which will ultimately lead to a better understanding of the mechanism of gene transcription as a whole.

Synthesis and characterization of a sulfur-containing phthalocyanine-gold nanoparticle hybrid

2015 ◽  
Vol 19 (01-03) ◽  
pp. 335-343 ◽  
Author(s):  
Vicente M. Blas-Ferrando ◽  
Javier Ortiz ◽  
Fernando Fernández-Lázaro ◽  
Ángela Sastre-Santos
Keyword(s):  
Gold Nanoparticle ◽  
Lipoic Acid ◽  
Zinc Phthalocyanine ◽  
Synthesis And Characterization ◽  
Ligand Exchange Reaction ◽  
H Nmr ◽  
Fluorescence Measurements ◽  
Transmission Electron ◽  
Yield Values

This work reports on the synthesis and characterization of a new gold nanoparticle-zinc phthalocyanine system, AuNP - S (t Bu )3 ZnPc , prepared by a ligand exchange reaction of tetraoctylammonium bromide with a novel unsymmetrically substituted zinc phthalocyanine which contains one thioester group in the peripheral position [ AcS (t Bu )3 ZnPc ]. The AuNP - S (t Bu )3 ZnPc hybrid was characterized using UV-vis and 1 H NMR spectroscopies. Transmission electron microscopy allowed the estimation of the size, which was calculated to be ~5 nm. AuNPs - S (t Bu )3 ZnPc conjugate showed much lower fluorescence quantum yield values than the AcS (t Bu )3 ZnPc demonstrating either an energy or electron transfer from the ZnPc to the AuNP . The AuNP - S (t Bu )3 ZnPc hybrid has been anchored to a TiO 2 semiconducting layer using lipoic acid. A solid configuration of TiO 2-lipoic acid- AuNP - S (t Bu )3 ZnPc has been prepared by anchoring lipoic acid to the TiO 2 ( TiO 2-LA) and introducing later the TiO 2-LA with free thiol groups in a toluene solution of AuNP - S (t Bu )3 ZnPc . We have also observed by UV-vis and fluorescence measurements the importance of the ZnPc in avoiding AuNP aggregation on the TiO 2 surface.

Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence

2021 ◽  
Author(s):  
Lok Man ◽  
William P. Klare ◽  
Ashleigh L. Dale ◽  
Joel A. Cain ◽  
Stuart J. Cordwell
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Lipid A ◽  
Phenotypic Characterization ◽  
Post Translational Modification ◽  
Form Of Life ◽  
Antimicrobial Interventions

Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation; with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.

Structure and Post-Translational Modification of the Lipoyl Domain of 2-Oxo Acid Dehydrogenase Complexes: A New Family of Protein Domains

1993 ◽  
pp. 283-288
Author(s):  
Richard N. Perham ◽  
Nicola G. Wallis ◽  
Simon M. Brocklehurst ◽  
Frederic Dardel ◽  
Adrian L. Davis ◽  
...  
Keyword(s):  
Protein Domains ◽  
Post Translational Modification ◽  
Acid Dehydrogenase ◽  
New Family ◽  
Lipoyl Domain

scholarly journals Non3 is an essential Drosophila gene required for proper nucleolus assembly

2019 ◽  
Vol 23 (2) ◽  
pp. 190-198
Author(s):  
E. N. Andreyeva ◽  
A. A. Ogienko ◽  
A. A. Yushkova ◽  
J. V. Popova ◽  
G. A. Pavlova ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Large Deletion ◽  
Protein Product ◽  
P Element ◽  
Post Translational Modification ◽  
Allelic Series ◽  
Larval Brain ◽  
Functional Studies ◽  
Mild Reduction

The nucleolus is a dynamic non-membrane-bound nuclear organelle, which plays key roles not only in ribosome biogenesis but also in many other cellular processes. Consistent with its multiple functions, the nucleolus has been implicated in many human diseases, including cancer and degenerative pathologies of the nervous system and heart. Here, we report the characterization of the Drosophila Non3 (Novel nucleolar protein 3) gene, which encodes a protein homologous to the human Brix domain-containing Rpf2 that has been shown to control ribosomal RNA (rRNA) processing. We used imprecise P-element excision to generate four new mutant alleles in the Non3 gene. Complementation and phenotypic analyses showed that these Non3 mutations can be arranged in an allelic series that includes both viable and lethal alleles. The strongest lethal allele (Non3∆600) is a genetically null allele that carries a large deletion of the gene and exhibits early lethality when homozygous. Flies heterozygous for Non3∆600 occasionally exhibit a mild reduction in the bristle size, but develop normally and are fertile. However, heteroallelic combinations of viable Non3 mutations (Non3197, Non3310 and Non3259) display a Minute-like phenotype, consisting in delayed development and short and thin bristles, suggesting that they are defective in ribosome biogenesis. We also demonstrate that the Non3 protein localizes to the nucleolus of larval brain cells and it is required for proper nucleolar localization of Fibrillarin, a protein important for post-translational modification and processing of rRNAs. In summary, we generated a number of genetic and biochemical tools that were exploited for an initial characterization of Non3, and will be instrumental for future functional studies on this gene and its protein product.

scholarly journals Characterization of The Interaction Between RIN13 (RPM1-Interacting13) and Sumo Proteins in Arabidopsis Thaliana

2017 ◽  
Vol 9 (2) ◽  
Author(s):  
Venny Santosa ◽  
Mio Nagabuchi ◽  
Sachiko Okada ◽  
Katsunori Tanaka
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescence Microscopy ◽  
Hypersensitive Response ◽  
Stress Responses ◽  
Molecular Interaction ◽  
Nuclear Body ◽  
Chromosome Organization ◽  
Post Translational Modification ◽  
Molecular Studies

<p>Small Ubiquitin-related MOdifier (SUMO) proteins can be found in many organisms, including <em>A. thaliana</em>, which possesses 9 SUMO genes. SUMO binds to various target proteins in a reversible reaction called SUMOylation. SUMOylation participates in transcription, chromosome organization, proteins localizations and stress responses. Our study showed that RIN13 (<span style="text-decoration: underline;">R</span>PM1-<span style="text-decoration: underline;">In</span>teracting<span style="text-decoration: underline;">13</span>/At2g20310) is a target of SUMOylation, which was initially found by interaction between this protein and AtSCE1a (E2). Recent report showed that overexpression of RIN13 enhanced the resistance to pathogen without inducing hypersensitive response. However, the molecular interaction between RIN13 and SUMO proteins and its significance have not been studied yet. Thus, our study aimed to characterize the interaction between RIN13 and SUMO proteins in <em>A. thaliana</em>. The result showed an isoform-specific SUMOylation between RIN13 and SUMO proteins. RIN13 is SUMOylated by SUMO1, 2, 3, and 5. Though expressed ubiquitously in <em>A.thaliana</em>, fluorescence microscopy showed that RIN13 localizes subcellularly in the nuclear body. Moreover, complete abolishment of SUMOylation with inactive E2 suggests the exclusion of RIN13 from nuclear body. These results showed that SUMOylation affected RIN13 localization, and indirectly influenced its interaction to other proteins and putative function. This paper presents evidence of RIN13 SUMOylation. Furthermore, RIN13 function in pathogenic resistance is shown to be supported by SUMOylation. Thus, this study enhanced the understanding of SUMO in plants and served as reference to molecular studies concerning post-translational modification of SUMO.</p>

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