Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase

Hecht, Nir; Regev, Ofir; Dovrat, Daniel; Aharoni, Amir; Gur, Eyal

doi:10.1016/j.jmb.2018.01.009

Cited by 8 publications

(11 citation statements)

References 35 publications

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“…Pup-Fl was purified as described by Hecht and Gur [27]. Pupylated FabD, PanB, and IdeR were expressed and purified as described by Hecht et al [25].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

et al. 2020

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Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a Pup-proteasome system tags target proteins for degradation by a bacterial proteasome. Pup, a small modifier protein, is attached to target proteins by PafA, the sole Pup ligase, in a process termed pupylation. In mycobacteria, including Mycobacterium smegmatis and Mycobacterium tuberculosis, Pup undergoes a deamidation step by the enzyme Dop prior to its PafA-mediated attachment to a target. The catalytic mechanism of Pup deamidation is also used by Dop to perform depupylation, namely the removal of Pup from already tagged proteins. Hence, Dop appears to play contradictory roles: On the one hand, deamidation of Pup promotes pupylation, while on the other hand, depupylation reduces tagged protein levels. To avoid futile pupylation-depupylation cycles, Dop activity must be regulated. An intramolecular regulatory mechanism directs Dop to catalyze deamidation more effectively than depupylation. A complementary intermolecular mechanism results in Dop depletion under conditions where protein pupylation and degradation are favorable. In this work, we studied these regulatory mechanisms and identified a flexible loop in Dop, previously termed the Dop-loop, that acts as an intramolecular regulatory element that allosterically controls substrate preference. To investigate regulation at the intermolecular level, we used the CRISPR interference system to knock down the expression of M. smegmatis ATP-dependent intracellular proteases and found that the ClpCP protease is responsible for Dop depletion under starvation conditions. These findings clarify previous observations and introduce a new level for the regulation of Dop activity.

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“…Pup-Fl was purified as described by Hecht and Gur [27]. Pupylated FabD, PanB, and IdeR were expressed and purified as described by Hecht et al [25].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

et al. 2020

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“…2011 ; Bolten et al. 2017 ; Hecht et al. 2018 ), we hypothesize that in the first reaction step, it catalyzed the formation of an acyl-Pup intermediate using Pup Q and ATP, as does wild type Dop.…”

Section: Resultsmentioning

confidence: 99%

Exploring Protein Space: From Hydrolase to Ligase by Substitution

Hecht

Monteil

Perrière

et al. 2020

Molecular Biology and Evolution

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The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

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“…We continued to investigate the role of the 3,4,5-trisubstitution on R 1 with R 2 being 3,4dihydroxyphenyl, like in the original two hits (entries [13][14][15][16][17][18][19][20][21][22][23][24][25]. This change of R 2 should deliver more potent compounds compared to the previous series.…”

Section: Resultsmentioning

confidence: 99%

“…This indicates that both the 3-methoxy and 4-hydroxy are essential for potent inhibitors. We thus continued investigating the effect of different substituents on the 5-position (entries [19][20][21][22][23][24].…”

Section: Resultsmentioning

confidence: 99%

“…It was however, not beneficial for the potency to add a more electron poor 4-fluorophenyl on position 5 (10g, entry 21). To further investigate the electronic properties, we tested analogs containing a Cl, Br or F on the 5position (10h-10j, entries [22][23][24]. 10h and 10i were slightly less potent than the original hit 1 The hybrid compound (10k) based on both 1 and 2 that has the 3-methoxy moiety replaced for a hydroxy moiety, was the most potent compound from the synthesized analogs (0.29 M, entry 25).…”

Section: Resultsmentioning

confidence: 99%

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Discovery and Optimization of Inhibitors for the Pup Proteasome System in Mycobacterium tuberculosis

Janssen

Zhang

Merkx

et al. 2019

Preprint

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Tuberculosis is a global health problem with the existence and spreading of multidrug resistant and extensive drug resistant strains. The development of new drugs for tuberculosis that inhibit different activities than the current drugs is thus urgent. The prokaryotic ubiquitin like protein proteasome system is an attractive target for the development of new drugs. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, a depupylase, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG53 (2). The hits were validated and determined to be fast reversible non-ATP competitive inhibitors. The SAR was established by testing 27 synthesized analogs of 1 and 2. Several of the synthesized compounds also inhibited the depupylation of a native substrate, FabD∼Pup. Importantly, the pupylation and depupylation activities of PafA, the sole Pup ligase in M. tuberculosis, was also inhibited by some of these compounds. With the identification of the first described lead compounds for Dop and PafA inhibition, this study shows that high throughput screening can be a successful strategy for this purpose.

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Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase

Cited by 8 publications

References 35 publications

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Exploring Protein Space: From Hydrolase to Ligase by Substitution

Discovery and Optimization of Inhibitors for the Pup Proteasome System in Mycobacterium tuberculosis

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