Targets of ubiquitin like system in mycobacteria and related actinobacterial species.

Protein turnover and recycling is a prerequisite in all living organisms to maintain normal cellular physiology. Many bacteria are proteasome deficient but they possess typical protease enzymes for carrying out protein turnover. However, several groups of actinobacteria such as mycobacteria harbor both proteasome and proteases. In these bacteria, for cellular protein turnover the target proteins undergo post-translational modification referred as pupylation in which a small protein Pup (prokaryotic ubiquitin-like protein) is tagged to the specific lysine residues of the target proteins and after that those target proteins undergo proteasomal degradation. Thus, Pup serves as a degradation signal, helps in directing proteins toward the bacterial proteasome for a turnover. Although the Pup-proteasome system has a multifaceted role in environmental stresses, pathogenicity and regulation of cellular signaling, but the fate of all types of pupylation such as mono and polypupylation on the proteins is still not completely understood. In this review, we present the mechanisms involved in the activation and conjugation of Pup to the target proteins, describing the structural sketch of pupylation and fundamental differences between the eukaryotic ubiquitin-proteasome and bacterial Pup-proteasome systems. We are also presenting a concise classification and cataloging of the complete battery of experimentally identified Pup-substrates from various species of actinobacteria.

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes.

Prokaryotic ubiquitin-like protein (Pup) and the modification enzymes involved in attaching Pup to or removing it from target proteins present a fascinating example of convergent evolution with respect to eukaryotic ubiquitination. Like ubiquitin (Ub), Pup is a small protein that can be covalently attached to lysine side chains of cellular proteins, and like Ub, it can serve to recruit tagged proteins for proteasomal degradation. However, unlike Ub, Pup is conformationally highly dynamic, exhibits a different linkage connectivity to its target lysines, and its ligase belongs to a different class of enzymes than the E1/E2/E3 cascade of ubiquitination. A specific feature of actinobacteria (aside from sporadic cases in a few other lineages), pupylation appears to have evolved to provide an advantage to the bacteria under certain environmental stresses rather than act as a constitutive modification. For Mycobacterium tuberculosis, pupylation and the recruitment of pupylated substrates to the proteasome support persistence inside host macrophages during pathogenesis, rendering the Pup-proteasome system an attractive drug target. In this review, we consider the dynamic nature of Pup in relation to its function, discuss the reaction mechanisms of ligation to substrates and cleavage from pupylated substrates, and put them in context of the evolutionary history of this post-translational modification.

Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins.

BACKGROUND: The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate. RESULTS: Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate. CONCLUSION: When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.

Effects of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on the mono-resistant to rifampin resistance to Mycobacterium tuberculosis / 中国人兽共患病学报

We studied the effect of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on mono-resistant to rifampin resistance to M.tuberculosis.A resazurin-based assay was employed to evaluate minimum inhibitory concentration (MIC) and comparative research on mono-resistant to rifampin MTB with Pup,Dop,PafA,Mpa genes expression and deletion of the difference.Above testing strains,respectively,carbonyl cyanide chlorobenzene hydrazone (CCCP),reserpine (RP),verapamil (VP)and chlorpromazine (CPZ) were tested.We compared and analyzed the change of rifampicin MICs on the various strains.Compared with rifampin resistant MTB,overexpression of Pup,Dop,PafA and Mpa genes were able to make monorifampicin of M.tuberculosis to enhance resistance to rifampin.Deletion of Pup gene,Mpa gene,Dop gene,PafA gene significantly decreased the resistance to rifampicin alone MTB,and the P value was ＜0.05.Results indicated that 4 kinds of efflux pump inhibitors can reduce the degree of rifampin MIC in different strains.Through the factorial analysis,there were some interactions between MTB and PPS efflux pump inhibitors,and the P value was ＜0.05.MTB PPS has influence on mono-rifampin resistance to MTB and it may regulate the efflux pathway related protein to influence its resistance.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein.

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy. To facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Förster resonance energy transfer (TR-FRET)-based assay for Dop function. The TR-FRET assay was successfully applied to determine the Michaelis constant for adenosine triphosphate (ATP) binding and to test the cofactor tolerance of Dop.

Correlation study between prokaryotic ubiquitin-like protein (Pup)-proteasome system of Mycobacterium tuberculosis and persistence of Mycobacterium tuber-culosis / 中国免疫学杂志

Objective:To explore the regulation of prokaryotic ubiquitin-like protein ( Pup )-proteasome system on the persistence of Mycobacterium tuberculosis by inducing Mycobacterium tuberculosis to being persistence state under hypoxia conditions and then analyzing the difference on the expression levels of Pup,Dop,PafA and Mpa gene at various time and different conditions. Methods:The total mRNA of the international standard virulent strains of Mycobacterium tuberculosis(H37Rv),which were cultured under hypoxia and aerobic conditions, were extracted from each group at various time and purity of the mRNA were identified.The expression of Pup,Dop,PafA and Mpa genes of M.tuberculosis strains in each group were quantified by SYBR Green I qRT-PCR,which aimed at finding the difference among the expression of Pup,Dop,PafA and Mpa genes at various time and different conditions.Results:The expression levels of Pup, Dop, PafA and Mpa genes in Mycobacterium tuberculosis under hypoxia conditions were measured at various times.The expression levels of Pup,Dop,PafA and Mpa genes:compared with the 0 d,the expression of the Pup gene was up-regulated 1.66,2.43 and 2.76-fold at 4 d,7 d,10 d respectively(P<0.05);the expression of the Dop gene was up-regulated 1.38, 1.91,2.54 and 3.28-fold at 2 d,4 d,7 d,10 d respectively(P<0.05);the expression of the PafA gene was up-regulated 1.22,1.75, 2.37 and 2.67-fold at 2 d,4 d,7 d,10 d respectively( P<0.05);the expression of the Mpa gene was up-regulated 1.66,2.21 and 2.63-fold at 4 d,7 d,10 d respectively(P<0.05).Take the aerobic conditions as control,under hypoxic conditions with the same culture time,the expression of the Pup gene was up-regulated 1.85,2.81 and 2.93-fold in 4 d,7 d,10 d respectively(P<0.05);the ex-pression of the Dop gene was up-regulated 1.20,1.76,2.01 and 3.01-fold in 2 d,4 d,7 d,10 d,respectively( P<0.05);the expression of the PafA gene was up-regulated 1.22,1.57,2.29 and 2.29-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05);the expression of the Mpa gene was up-regulated 1.16,1.58,2.16 and 2.69-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05).Conclusion:Under hypoxic conditions,there were significant differences on the expressions of Pup, Dop, PafA and Mpa genes at various times;what′s more, significant differences on the expressions of Pup,Dop,PafA and Mpa genes exist between hypoxic and aerobic conditions at the same time,Prokaryotic ubiquitin-like protein( Pup)-proteasome system plays a regulatory role on M.tuberculosis′s persistence.

Effects of the Pup-proteasome system on the growth of Mycobacterium smegmatis strains / 中华微生物学和免疫学杂志

Objective To study the effects of prokaryotic ubiquitin-like protein ( Pup)-proteasome system on the growth of Mycobacterium strains.Methods The genes encoding Pup ( pup gene) and protea-someβsubunit ( prcB gene) were respectively knocked out from Mycobacterium smegmatis ( M.sm) strains by homologous recombination.The growth and viability of the wild-type and mutant strains of M.sm were an-alyzed under normal culture condition and under hypoxia as well as anaerobic conditions.Results The pup and prcB genes were completely and precisely knocked out from M.sm strains and the mutant strains were named △SM-Pup and△SM-prcB, respectively.The△SM-Pup strains grew faster than the wild type ( WT) and△SM-prcB strains.No significantly differences in the growth of M.sm were found between the WT and△SM-prcB strains.Conclusion The Pup-proteasome system was involved in the growth of M.sm, espe-cially the pup gene.There was difference between pup and prcB genes in regulating the growth of M.sm.The functions and influences of Pup-proteasome system still need further investigation.

Pupylation as a signal for proteasomal degradation in bacteria.

Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that certain bacteria use a tagging pathway functionally related to ubiquitination in order to target proteins for proteasomal degradation. However, functional analogies do not translate into structural or mechanistic relatedness. Bacterial Pup, unlike eukaryotic Ub, does not adopt a ß-grasp fold, but is intrinsically disordered. Furthermore, isopeptide bond formation in the pupylation process is carried out by enzymes evolutionary descendent from glutamine synthetases. While in eukaryotes, the proteasome is the main energy-dependent protein degradation machine, bacterial proteasomes exist in addition to other architecturally related degradation complexes, and their specific role along with the role of pupylation is still poorly understood. In Mycobacterium tuberculosis (Mtb), the Pup-proteasome system contributes to pathogenicity by supporting the bacterium's persistence within host macrophages. Here, we describe the mechanism and structural framework of pupylation and the targeting of pupylated proteins to the proteasome complex. Particular attention is given to the comparison of the bacterial Pup-proteasome system and the eukaryotic ubiquitin-proteasome system. Furthermore, the involvement of pupylation and proteasomal degradation in Mtb pathogenesis is discussed together with efforts to establish the Pup-proteasome system as a drug target. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis.

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.

Correlation study between prokaryotic ubiquitin-like protein (Pup)-proteasome and drug resistance of clinical isolates of Mycobacterium tuberculosis / 中国免疫学杂志

Objective:To study the correlation different drug-resistant mycobacterium tuberculosis clinical isolates of prokaryotic ubiquitin-like protein ( Pup )-proteasome of Pup, Dop, PafA, Mpa gene expression level and mycobacterium tuberculosis Pup-proteasome system with Xinjiang region widely popular drug-resistant mycobacterium tuberculosis in clinical isolates resistance.Methods:Total RNA of Mtb was extracted from cultured Mtb during the logarithmic phase in drug-susceptible strains in Xinjiang region,the clinical strains drug sensitive to INH,RFP,SM and EB respectively,and multidrug-resistant(MDR) strains.And then the purity of total RNA was identified.The expressing of Pup-proteasome relevant gene( Pup,Dop,Mpa,PafA) were quantified using SYBR GreenⅠqRT-PCR which aimed at finding the correlation between Mtb Pup-proteasome system and drug resistance of Mtb clinical isolates widespread in Xingjiang region by analyzing the expression of Pup, Dop, PafA, Mpa gene among different isolates.Results:Compared with the drug-senstive clinical isolates, mRNA expression level of Pup, Mpa gene was down-regulated in resistant M.tuberculosis clinical isolates INH ( INH-MTB) ,RFP ( RFP-MTB) ,SM ( SM-MTB) and EB ( EB-MTB) ,mRNA expression levels of genes in Dop and PafA was higher in resistant M.tuberculosis clinical isolates,the difference was statistically significant(P<0.05).Compared with MDR strain, the expression of Pup, Dop, Mpa gene were up-regulated different in the resistant M.tuberculosis clinical isolates isolates ,the expression of PafA gene was down-regulated different,the difference was statistically significant( P<0.05).Conclusion:The differentially expressed gene of Pup、Dop、PafA、Mpa gene in sensitive strains,INH-MTB,RFP-MTB,SM-MTB,EB-MTB and MDR strains.The Mycobacterium tuberculosis Pup-proteasome system.Therefore,the Pup-proteasome system have association with the drug resistance of Mtb strains widespread in Xinjiang region.

Study progress of prokaryotic ubiquitin-like protein (Pup)-proteasome system of My cobacterium tuberculosis / 中国人兽共患病学报

Proteasome pathway is another major pathway of protein degradation in addition to lysosome in eukaryotic cell ,which involved a number of physiological functions regulation in cell .Prokaryotic ubiquitin-like protein was found in My-cobacterium tuberculosis in 2008 .With the effect of co-factor Dop ,PafA and Mpa ,Pup can mark a variety of protein ubiquitina-tion followed by importing them into proteasomal degradation .The target protein of Pup-proteasome system like FabD ,PanB , Ino1 ,Icl ,SodA ,and MtrA are involved with metabolism ,signal transduction pathways ,virulence factors ,pathogenicity and the persistence of bacteria in the host cell .Proteasome inhibitor make the function of proteasome restricted and the accumula-tion of Pup’s labeled substrate result in changes in the expression of gene indirectly ,which impacted the ability of resistance to outside pressure and the pathogenicity of Mycobacterium tuberculosis . The finding Pup-proteasome system reveals a novel mechanism of protein degradation in prokaryotes ,which is expected to become a new target of treatment of anti-TB drugs . Here ,we summarize the progress on the Pup-proteasome system in Mycobacterium tuberculosis .