AAA+ Machines of Protein Destruction in Mycobacteria

Alhuwaider, Adnan Ali H.; Dougan, David A.

doi:10.3389/fmolb.2017.00049

Cited by 32 publications

(36 citation statements)

References 98 publications

(142 reference statements)

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“…Other known AAA+ proteases in M. smegmatis include FtsH, Lon, ClpXP, and ClpCP. The latter two comprise a tetradecameric proteolytic core made of ClpP1 and ClpP2 proteins [28]. Examining Dop depletion in cells lacking ClpXP and ClpCP is complicated by the fact that these proteases are essential for M. smegmatis survival.…”

Section: Dop Degradation Is Clpcp-dependentmentioning

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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Section: Dop Degradation Is Clpcp-dependentmentioning

confidence: 99%

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

et al. 2020

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“…Excellent reviews have extensively explored drug discovery for the bacterial proteolysis machinery. 188 − 192 We have summarized information regarding inhibitors and activators of the Mtb proteolysis systems in Table 2 . Targeting bacterial proteolysis provides a unique situation in which either inhibiting or activating the target pathway results in bacterial death.…”

Section: Targeting the Proteostasis Network: Clpc1 Clpp1p2 And Mtb2mentioning

confidence: 99%

Targeting the Proteostasis Network for Mycobacterial Drug Discovery

et al. 2018

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world’s deadliest infectious diseases and urgently requires new antibiotics to treat drug-resistant strains and to decrease the duration of therapy. During infection, Mtb encounters numerous stresses associated with host immunity, including hypoxia, reactive oxygen and nitrogen species, mild acidity, nutrient starvation, and metal sequestration and intoxication. The Mtb proteostasis network, composed of chaperones, proteases, and a eukaryotic-like proteasome, provides protection from stresses and chemistries of host immunity by maintaining the integrity of the mycobacterial proteome. In this Review, we explore the proteostasis network as a noncanonical target for antibacterial drug discovery.

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“…Previous studies made use of a multi-pronged approach involving isothermal calorimetry, NMR spectroscopy, crystallography, and functional assays to understand the mechanism of allosteric activation of Thermus (13,14). This regulation is partly achieved through the binding of ClpC1 or ClpX regulatory particles, which is coupled to ClpP protease active sites (28,44). A second layer of protection against unwanted degradation is afforded by the modest affinities of MtClpP1 and MtClpP2 for each other, and the fact that individual ClpP rings are catalytically dead since interactions between Asp and Arg residues on adjacent rings are required to couple the oligomerization state of the enzyme to its catalytic activity.…”

Section: Generation Of a Catalytically Dead Enzyme By Substituting Acmentioning

confidence: 99%

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

Vahidi

Ripstein

Juravsky

et al. 2019

Preprint

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Short title: NMR and cryo-EM of M. tuberculosis ClpP1P2The MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB.Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health. the binding of essential AAA+ unfoldases, ClpX or ClpC1, that use the energy of ATP to unfold and translocate substrates into its catalytic chamber for degradation (28). Lassomycin (29), ecumicin (30), and rufomycin (31) are promising antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by binding to ClpC1 and decoupling ATP-dependant protein unfolding from proteolysis. Similarly, ADEPs also kill M. tuberculosis by preventing the binding of AAA+ regulatory unfoldases to MtClpP1P2 (32). MtClpP1P2 reacts with standard inhibitors of serine proteases, such as chloromethyl ketones, which modify the serine and histidine residues at enzyme active sites (33). Peptide boronates have been shown to also directly engage MtClpP1P2 active sites, causing inhibition at low micromolar concentrations and preventing growth of M. tuberculosis (34-36). More recently Cediranib, an anti-cancer drug, was proposed as a novel non-covalent inhibitor of MtClpP1P2 (37).Most bacteria possess a single clpP gene, giving rise to a structure comprising a pair of heptameric rings that are arranged coaxially to form a homotetradecameric barrel-like protein complex enclosing fourteen Asp-His-Ser catalytic triads (11, 12,38). Each of the identical protomers consists of an N-terminal domain that forms gated narrow pores on the apical surface of the barrel, a head domain that generates the main body of the ClpP barrel, and a handle region comprising a helix and a β-sheet that mediate ClpP ring-ring interactions (11,(38)(39)(40). In addition, the handle provides crucial contacts that align the catalytic triad and generates a binding grove for substrate polypeptides (41). Opening of the pores that allow substrate translocation is tightly regulated by AAA+ regulators (42,43). Actinobacteria, the phylum to which mycobacteria such as M. tuberculosis belong, are unique in that they contain two clpP genes, clpP1 and clpP2, that encode for MtClpP1 and MtClpP2, respectively (44,45). Initial structure-function studies concluded that MtClpP1 and SI AppendixAn allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR ...

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AAA+ Machines of Protein Destruction in Mycobacteria

Cited by 32 publications

References 98 publications

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Targeting the Proteostasis Network for Mycobacterial Drug Discovery

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

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