Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.     

Pup-蛋白酶体系统的作用机制和生物学功能

Pup-蛋白酶体系统（Pup-proteasome system,PPS）是原核生物的一种翻译后蛋白质修饰降解体系,在去酰胺酶（deamidase of Pup,Dop）和蛋白酶体辅助因子A (proteasome accessory factorA,PafA)两种酶的作用下,原核生物类泛素蛋白（prokaryotic ubiquitin-like protein,Pup）可以标记靶蛋白,并介导靶蛋白经蛋白酶体降解。在分枝杆菌中PPS参与氧化应激、营养缺乏、热激、DNA损伤等多种应激反应,并在金属离子稳态调控、毒素-抗毒素系统（toxin-antitoxin system,TA system）的调节以及抵抗宿主免疫等过程中发挥作用。PPS与结核分枝杆菌（Mycobacterium tuberculosis,Mtb）的持留性和致病性直接相关,因此PPS中的PafA、Dop和蛋白酶体均是抗结核药物开发的新靶点,筛选针对PPS的小分子抑制剂将成为新型抗结核药物研发的一个新途径。此外,Paf A催化的蛋白质Pup化被应用于生物技术的研发,形成了一种新的邻近标记技术——基于Pup化的邻近标记技术...     

Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

Dop functions as a depupylase in the prokaryotic ubiquitin‐like modification pathway

Post‐translational modification of proteins with prokaryotic ubiquitin‐like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two‐step process in which the carboxy‐terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ‐carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.     

The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. It has been reported that that M. smegmatis PafA can be poly-pupylated. In this study, the mechanism of PafA self-pupylation is explored. We found that K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in M. smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop. The polymeric Pup chain formed through K7/K31 linkage is much more sensitive to Dop than the mono-Pup directly attached to PafA. Moreover, self-pupylation of PafA is involved in the regulation of its stability in vivo in a proteasome-dependent manner, suggesting that PafA self-pupylation functions as a mechanism in the auto-regulation of the Pup-proteasome system.     

Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.     

Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination

   

Recently Mycobacterium tuberculosis was shown to possess a novel protein modification, in which a small protein Pup is conjugated to the epsilon-amino groups of lysines in target proteins. Analogous to ubiquitin modification in eukaryotes, this remarkable modification recruits proteins for degradation via archaeal-type proteasomes found in mycobacteria and allied actinobacteria. While a mycobacterial protein named PafA was found to be required for this conjugation reaction, its biochemical mechanism has not been elucidated. Using sensitive sequence profile comparison methods we establish that the PafA family proteins are related to the γ-glutamyl-cysteine synthetase and glutamine synthetase. Hence, we predict that PafA is the Pup ligase, which catalyzes the ATP-dependent ligation of the terminal γ-carboxylate of glutamate to lysines, similar to the above enzymes. We further discovered that an ortholog of the eukaryotic PAC2 (e.g. cg2106) is often present in the vicinity of the actinobacterial Pup-proteasome gene neighborhoods and is likely to represent the ancestral proteasomal chaperone. Pup-conjugation is sporadically present outside the actinobacteria in certain lineages, such as verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes, and in the latter two lineages it might modify membrane proteins.     

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.     

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli

Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium-specific cofactors for substrate recognition by PafA in vivo.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Activity of the mycobacterial proteasomal ATPase Mpa is reversibly regulated by pupylation

Pupylation is a bacterial post-translational modification of target proteins on lysine residues with prokaryotic ubiquitin-like protein Pup. Pup-tagged substrates are recognized by a proteasome-interacting ATPase termed Mpa in Mycobacterium tuberculosis. Mpa unfolds pupylated substrates and threads them into the proteasome core particle for degradation. Interestingly, Mpa itself is also a pupylation target. Here, we show that the Pup ligase PafA predominantly produces monopupylated Mpa modified homogeneously on a single lysine residue within its C-terminal region. We demonstrate that this modification renders Mpa functionally inactive. Pupylated Mpa can no longer support Pup-mediated proteasomal degradation due to its inability to associate with the proteasome core. Mpa is further inactivated by rapid Pup- and ATPase-driven deoligomerization of the hexameric Mpa ring. We show that pupylation of Mpa is chemically and functionally reversible. Mpa regains its enzymatic activity upon depupylation by the depupylase Dop, affording a rapid and reversible activity control over Mpa function.     

Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase.

We report the isolation and characterization of a Neurospora crassa glutamine synthetase (GS) mutant altered in one of the two polypeptides (GS alpha) of this enzyme. We used the gln-1bR8 mutant strain that synthesizes only the GS alpha monomer and lacks the GS beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-DL-methionine-SR-sulfoximine (alpha-me-MSO), an inhibitor of GS activity. The GS activity of the gln-1bR8;alpha-me-MSOR strain drastically reduced its transferase activity and only slightly reduced its synthetase activity, and it was resistant to inhibition by alpha-me-MSO and L-methionine-DL-sulfoximine. The mutation that overcame the inhibitory effect of alpha-me-MSO also altered the antigenic, kinetic, and physical properties of GS alpha. The low GS activity of the alpha-me-MSO-resistant strain was compensated for by a higher glutamate/glutamine ratio and a lower glutamate synthase activity, allowing this strain to grow as well as the parental strain. The mutation that conferred resistance to alpha-me-MSO was not linked to the gln-1bR8 mutation, providing direct evidence of the existence of two genes involved with the structure of the two polypeptides of N. crassa GS.     

Isolation of a New Tobacco Constituent, 5,6-Dihydro-5-hydroxy-3,6-epoxy-β-ionol,from Japanese Domestic Suifu Tobacco

Glutamine production was investigated by coupling of glutamine synthetase from Gluconobacter suboxydans with a sugar fermentation system of baker's yeast (energy generating system). Under the optimum condition, 22 mM glutamine was formed in 3 hr, and the yield was 92% based on the substrate glutamate. The first step of the process was the accumulation of fructose 1,6-diphosphate (FDP) as a reservoir of fermentation energy, in the presence of a high concentration of inorganic phosphate; and the second step was accomplished by coupling the degradation of FDP with glutamine synthetase reaction through an ADP-ATP system. The effects of enzyme concentration, additives in the reaction mixture and others on glutamine formation were investigated, and the importance of three factors was pointed out: (a) the ratio of activity of energy generating system to utilizing system, (b) contaminated enzyme(s) in the energy utilizing system and (c) the enzymatic properties of the energy utilizing system.     

Glutamine synthetase of Chlamydomonas: Rapid reversible deactivation

A 70% reduction in glutamine synthetase (GS) activity was observed within 5 min when 5 mM NH₃ and darkness was applied to steady-state cells of Chlamydomonas utilising NO₃. The enzyme was reactivated in vivo by reillumination of the culture and in vitro by treatment with thiol reagents. The activity modulations affected the synthetase and transferase activities similarly and were not influenced by protein synthesis inhibitors. Deactivation of GS was also observed when steady-state cells were treated with an uncoupler of phosphorylation, carbonylcyanide m-chlorophenylhydrazone (CCCP) or inhibitors of the electron transport chain but under these conditions the activity modulation affected over 90% of the activity and was irreversible. The mechanism of the physiological deactivation of GS is discussed in relation to both the in vivo and in vitro findings.Abbreviations GS glutamine synthetase (EC 6.3.1.2.) - GSs glutamine synthetase, synthetase activity - GSt glutamine synthetase, transferase activity - CAP chloramphenicol - CCCP carbonylcyanide m-chlorophenyl hydrazone - CHX cycloheximide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DSPD disalicylidene propanediamine - DTT dithiothreitol - GSH reduced glutathione     

In vivo regulation of glutamine synthetase activity in the marine chlorophyll b-containing cyanobacterium Prochlorococcus sp. strain PCC 9511 (oxyphotobacteria)

The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.     

Effect of lead on nitrogenase and enzymes of nitrogen assimilation in a cyanobacterium Nostoc muscorum.

Lead decreased the growth rates, total cell mass, heterocyst frequency, total cell protein, nitrogenase activity, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in N:muscorum. However, lead at 0.01 and 10 micrograms ml-1 conc. enhanced nitrogenase as well as GS activity of the cells. On transfer to excess lead (100 micrograms ml-1), nitrogenase and GS activities ceased almost after 24 hr in the cyanobacterium. It is deduced that lead has a two step effect on stimulation and inhibition of metabolic activity at 0.01 and 10 micrograms ml-1 concentration and 0.1 and 100 micrograms ml-1 concentration respectively indicating a close interaction between nitrogen fixation and GS activity. However, GOGAT activity is an exception to this two step stimulation and inhibition process.     

Regulation of glutamine synthetase activity and synthesis in free-living and symbiotic Anabaena spp.

Regulation of the synthesis and activity of glutamine synthetase (GS) in the cyanobacterium Anabaena sp. strain 7120 was studied by determining GS transferase activity and GS antigen concentration under a variety of conditions. Extracts prepared from cells growing exponentially on a medium supplemented with combined nitrogen had a GS activity of 17 mumol of gamma-glutamyl transferase activity per min per mg of protein at 37 degrees C. This activity doubled in 12 h after transfer of cells to a nitrogen-free medium, corresponding to the time required for heterocyst differentiation and the start of nitrogen fixation. Addition of NH3 to a culture 11 h after an inducing transfer immediately blocked the increase in GS activity. In the Enterobacteriaceae, addition of NH3 after induction results in the covalent modification of GS by adenylylation. The GS of Anabaena is not adenylylated by such a protocol, as shown by the resistance of the transferase activity of the enzyme to inhibition by Mg2+ and by the failure of the enzyme to incorporate 32P after NH3 upshift. Methionine sulfoximine inhibited Anabaena GS activity rapidly and irreversibly in vivo. After the addition of methionine sulfoximine to Anabaena, the level of GS antigen neither increased nor decreased, indicating that Glutamine cannot be the only small molecule capable of regulating GS synthesis. Methionine sulfoximine permitted heterocyst differentiation and nitrogenase induction to escape repression by NH3. Nitrogen-fixing cultures treated with methionine sulfoximine excreted NH3. The fern Azolla caroliniana contains an Anabaena species living in symbiotic association. The Anabaena species carries out nitrogen fixation sufficient to satisfy all of the combined nitrogen requirements of the host fern. Experiments by other workers have shown that the activity of GS in the symbiont is significantly lower than the activity of GS in free-living Anabaena. Using a sensitive radioimmune assay and a normalization procedure based on the content of diaminopimelic acid, a component unique to the symbiont, we found that the level of GS antigen in the symbiont was about 5% of the level in free-living Anabaena cells. Thus, the host fern appears to repress synthesis of Anabaena GS in the symbiotic association.     

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.