ADEP1 activated ClpP1P2 macromolecule ofLeptospira, an ideal Achilles’ heel to deregulate proteostasis and hamper the cell survival

Abstract

The caseinolytic protease (ClpP) complex in Leptospira interrogans is unusual in its functional activation. The genus Leptospira has two ClpPs, ClpP1 and ClpP2, which transcribes independently, regardless it couples to form the active tetradecamer. Acyldepsipeptide (ADEP) antibiotic hampers the growth of numerous bacterial species by activating the target protein ClpP and dysregulating the physiological proteostasis within the cell. In vitro culture of the L. interrogans fortified with the ADEP impeded the spirochete growth accompanied by a more elongated morphology. The chemoactivation of the ClpP is conditional on the duration of the self-compartmentalization of each of the ClpP isoforms. The small extent (10 min) self-assembled ClpP1P2 revealed inhibition in the peptidase activity (7-fold) in the presence of the ADEP due to the self-cleavage of the ClpP subunits. On supplementation of the β-casein or bovine serum albumin, the peptidase activity of the ClpP1P2 (short-incubated) got enhanced by the ADEP, while the ClpP1P2 (long-incubated) activity was retained to the same level. ADEP can also switch on the ClpP1P2 from a strict peptidase into proteolytic machinery that discerns and degrades the unfolded protein substrates autonomous of the cognate chaperone ClpX. In consensus to the most prokaryotes with the multi ClpP variants, the computational prototype of the ClpP1P2 tertiary structure infers that the hydrophobic pocket wherein the ADEPs predominantly docks are present in the ClpP2 heptamer. Additionally, the dynamic light scattering and the site-directed mutagenesis of a catalytic serine residue in either of the ClpP isoforms proposes a second interaction site for the ADEP.

Trigger Factor in Association with the ClpP1P2 Heterocomplex of Leptospira Promotes Protease/Peptidase Activity

“… 40 Interestingly, in the presence of an adequate amount of substrate casein, such an autoproteolysis event of overactivated ADEP1-bound ClpP1P2 is reduced. 40 With this perception, it is currently critical to address if rLinTF acts as an additional substrate to the model casein for the ADEP-activated ClpP1P2 proteolytic chamber, resulting in further increase protease stimulation due to reduction of autoproteolysis. To address this, β-casein substrate degradation was analyzed by the ADEP1-bound rClpP1P2 heterocomplex activity at 30 min time intervals for 2 h in the presence and absence of rLinTF.…”

“… 37 − 39 In Leptospira , ADEP1-activated rClpP1P2 shows stimulated peptidase activity. 40 Next, in order to gain insights into the stimulation function of LinTF, we evaluated whether rLinTF can further unconditionally impact the ADEP1-bound rClpP1P2 peptidase activity. Thus, another peptidase assay was conducted using ADEP1-bound rClpP1P2 on model substrate S1 (Suc-LY-AMC) in the presence of rLinTF.…”

“…In Leptospira , antibiotic acyldepsipeptide (ADEP1) has been illustrated to activate the proteolytic activity of the ClpP1P2 heterocomplex to degrade protein substrates even in the absence of co-chaperone ClpX. 40 To gain insights into the novel function of stabilizing/generating the functional multimeric protein by the rLinTF, an identical protease assay was conducted with the ADEP1-bound rClpP1P2 in the presence of rLinTF. Protease activity of ADEP1-bound rClpP1P2 was found to be further enhanced (∼3-fold) on supplementation of rLinTF in the reaction mixture versus its control with no rLinTF at 2 h of the reaction period ( Figure 3 B).…”

“…ADEP1 is a natural antibiotic belonging to the class of acyldepsipeptides , which acts by dysregulating the activity of ClpP protease. − In Leptospira, ADEP1-activated rClpP1P2 shows stimulated peptidase activity . Next, in order to gain insights into the stimulation function of LinTF, we evaluated whether rLinTF can further unconditionally impact the ADEP1-bound rClpP1P2 peptidase activity.…”

“…In Leptospira, antibiotic acyldepsipeptide (ADEP1) has been illustrated to activate the proteolytic activity of the ClpP1P2 heterocomplex to degrade protein substrates even in the absence of co-chaperone ClpX . To gain insights into the novel function of stabilizing/generating the functional multimeric protein by the rLinTF, an identical protease assay was conducted with the ADEP1-bound rClpP1P2 in the presence of rLinTF.…”