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co-translational transport, and post-translational modification

In S. cerevisiae, there are two thiouridines in tRNA, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) in cytosolic tRNAs and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U34) in mitochondrial tRNAs. The biosynthesis pathway of 2-thiouridine in cytosolic tRNA is Fe–S cluster dependent, while the mitochondrial pathway is independent of Fe–S cluster formation (; ). The biosynthesis of s2U in cytosolic tRNA in the eukaryote utilizes a protein-thiocarboxylate as intermediate sulfur donor. This pathway is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes and a similar biosynthesis pathway in archaea was reported (; ).

A characteristic structural and functional feature of RNA is post-transcriptional modification. More than 100 forms of naturally occurring chemical modification have been reported to date, (; ). The roles of modified nucleosides in tRNA are important and wide-ranging, and include critical roles in biogenesis, structural stability, codon recognition, maintenance of reading frame, and identification elements for the translation machinery (; ).

POST-TRANSLATIONAL MODIFICATION OF CELLULAR ..

Keywords: Arabidopsis, Post-translational Modification, ..

formed during post-translational protein modification of proline by ..

Quantification of Lal formation after alkaline treatment by GC–MS has been challenging because of the large background resulting from the leader peptide and the biosynthetic enzymes. In an effort to improve both the amounts of material and their purity, we turned to coexpression of the CinA peptide and the CinM and CinX modification enzymes in E. coli. Recent studies in several laboratories have demonstrated the feasibility of conducting the post-translational modifications that generate lantibiotics in this heterologous host. In our laboratory, pRSFDuet-1 and pACYCDuet-1 plasmids were used for these studies, which offer convenient purification of the post-translationally modified peptide using IMAC because of a His6-tag at the N-terminus of the leader peptide of the substrate peptide.() A similar approach was employed here in an attempt to obtain higher quantities of peptide processed by CinM and CinX.

The order in which CinM and CinX may carry out their post-translational modifications was also investigated. In vitro these two enzymes did not have a compulsory order of action as His10-CinM was able to dehydrate and cyclize His6-CinA that had been hydroxylated by His6-CinX (), and His6-CinX was able to hydroxylate His6-CinA that had been dehydrated and cyclized (Figure ). To validate the site of hydroxylation, aspartate 15 was mutated to alanine, and the mutant CinA peptide was incubated with His6-CinX. MALDI ToF MS analysis showed no change in the mass of the mutant peptide (data not shown). In lantibiotics, the leader peptide is required for activity of most of the biosynthetic enzymes.() To evaluate if this requirement was also the case for His10-CinM and His6-CinX, the core peptide of CinA (CinA1–19) was synthesized by solid-phase peptide synthesis. As expected, His10-CinM showed no activity in the absence of the leader peptide (). In contrast, His6-CinX was capable of hydroxylating CinA(1–19) (). LC–MS/MS analysis of the product further verified that the hydroxylation occurs on Asp15 ().

autocatalytic post-translational modification to form a ..

Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A). Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the biosynthesis of these sulfur modifications starts with cysteine desulfurases, which catalyze the generation of persulfide (an activated form of sulfur) from cysteine. Many sulfur-carrier proteins are responsible for delivering this activated sulfur to each biosynthesis pathway. Finally, specific “modification enzymes” activate target tRNAs and then incorporate sulfur atoms. Intriguingly, the biosynthesis of 2-thiouridine in all domains of life is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes. This review summarizes the recent characterization of the biosynthesis of sulfur modifications in tRNA and the novel roles of this modification in cellular functions in various model organisms, with a special emphasis on 2-thiouridine derivatives. Each biosynthesis pathway of sulfur-containing molecules is mutually modulated via sulfur trafficking, and 2-thiouridine and codon usage bias have been proposed to control the translation of specific genes.

Prediction and Analysis of Post-Translational Pyruvoyl Residue Modification ..

Unexpectedly, the characterization of the biosynthesis of 2-thiouridine has revealed molecular fossils, namely, ancient ubiquitin-like molecules, including Urm1 in eukaryotes, TtuB in bacteria, and SAMP2 in archaea. These proteins have two functions; they function as sulfur carriers for 2-thiouridine synthesis and as protein modifiers. Therefore, these proteins may be evolutionarily intermediates between ancient sulfur-carrier proteins and protein modifiers. It is possible that an adenylated or thiocarboxylated intermediate, formed in the course of 2-thiouridine biosynthesis, was incidentally attached to adjacent proteins at some time in the past. By this post-translational modification, the activities of the attached proteins have probably changed. This was certainly the origin of the post-translational modification of proteins by these Ubls. The primitive function of these conjugates was probably self-regulation. Conjugates of Ubls and the modification enzymes Ncs6/Ncs2 and TtuA have already been detected, but the function of these post-translational modifications remains to be clarified. The resultant conjugates have become to be used as tags for the recognition and regulation of other proteins. By acquiring E2 and E3 enzymes, which can recognize target proteins precisely, the Ub system evolved considerably to become the sophisticated system it is today in eukaryotes.

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of Post-translational Modifications of Plant Protein ..


and post-translational modification

The mobilization of sulfur in biosynthesis pathways of sulfur-containing compounds starts with the activation of the sulfur atom of cysteine by the cysteine desulfurase IscS. IscS forms an enzyme-bound persulfide and this activated sulfur is transferred to the next acceptor protein in each pathway, such as TusA (2-thiouridine in tRNA), ThiI (4-thiouridine in tRNA), IscU (Fe–S cluster), ThiS (thiamin), and MoaD (molybdenum cofactor; Figure ). It is conceivable that each biosynthesis pathway of sulfur-containing molecules is mutually modulated via competition of sulfur trafficking. An interesting observation with respect this was made during a study of lambda phage infection in E. coli (, ). During viral infection, the normal amount of modified uridine in tRNALysUUU grarantees a normal translation and frameshifting rate for production of the proper ratio of viral gpG and gpGT proteins (gpGT production needs programmed ribosomal frameshifting). Hypomodification of tRNALysUUUcaused by deletion of Tus genes in the host cell leads to increased frameshifting in the translation of viral mRNA of G and T genes, which affects the ratio of viral gpG to gpGT. A lower gpG:gpGT ratio leads to decreased virion production. Another factor lowering infection is overexpression of IscU in the host cell. In this situation, higher sulfur flow from IscS to IscU conversely lowers the sulfur flow to Tus proteins, which leads to hypomodification of tRNALysUUU and abnormal frameshifting, which finally affects the viral infection rate. The competitive binding of TusA and IscU to IscS has been analyzed in detail, based on the structures of the complexes IscS/TusA and IscS/IscU (; ).

post-translational modification, in ..

CinX is shown to be an Fe(II)/α-ketoglutarate-dependent hydroxylase that catalyzes the β-hydroxylation of Asp15 in the CinA precursor peptide. Interestingly, His6-CinX accepted the core region of CinA as a substrate without the need of the leader sequence. Similar observations were made for the oxidative decarboxylase EpiD,() suggesting that tailoring enzymes that introduce post-translational modifications beyond dehydration and cyclization may not require the leader peptide. However, as observed for other lanthionine synthetases, His10-CinM required the leader peptide for efficient processing. Changing the order of incubation of the precursor peptide with His10-CinM or His6-CinX resulted in the same product.

The post-translational modification of arginine residues ..

In some thermophiles, 5-methyl-2-thiouridine (m5s2U) [also called 2-thioribothymidine (s2T)] occurs at position 54 in the T-loop (Figure ). Intriguingly, the biosynthesis pathway () is similar to that of cytosolic s2U34 in eukaryotes, and ubiquitin-like post-translational modification of cellular proteins has recently been discovered also in the bacteria domain ().

the enzyme’s secretion by post-translational modification

Benjdia A, Leprince J, Guillot A, Vaudry H, Rabot S, Berteau O; , J Am Chem Soc. 2007;129:3462-3463.: Anaerobic sulfatase-maturating enzymes: radical SAM enzymes able to catalyze in vitro sulfatase post-translational modification.

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