The Bacterial Proteasome at the Core of Diverse Degradation Pathways

Proteasomal protein degradation exists in mycobacteria and other actinobacteria, and expands their repertoire of compartmentalizing protein degradation pathways beyond the usual bacterial types. A product of horizontal gene transfer, bacterial proteasomes have evolved to support the organism's survival under challenging environmental conditions like nutrient starvation and physical or chemical stresses. Like the eukaryotic 20S proteasome, the bacterial core particle is gated and must associate with a regulator complex to form a fully active protease capable of recruiting and internalizing substrate proteins. By association with diverse regulator complexes that employ different recruitment strategies, the bacterial 20S core particle is able to act in different cellular degradation pathways. In association with the mycobacterial proteasomal ATPase Mpa, the proteasome degrades substrates post-translationally modified with prokaryotic, ubiquitin-like protein Pup in a process called pupylation. Upon interaction with the ATP-independent bacterial proteasome activator Bpa, poorly structured substrates are recruited for proteasomal degradation. A potential third degradation route might employ a Cdc48-like protein of actinobacteria (Cpa), for which interaction with the 20S core was recently demonstrated but no degradation substrates have been identified yet. The alternative interaction partners and wide range of substrate proteins suggest that the bacterial proteasome is a modular, functionally flexible and conditionally regulated degradation machine in bacteria that encounter rapidly changing and challenging conditions

Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

AbstractCell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins

Structure and functional implications of WYL domain-containing bacterial DNA damage response regulator PafBC

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.ISSN:2041-172

The Mycobacterial LexA/RecA-Independent DNA Damage Response Is Controlled by PafBC and the Pup-Proteasome System

Mycobacteria exhibit two DNA damage response pathways: the LexA/RecA-dependent SOS response and a LexA/RecA-independent pathway. Using a combination of transcriptomics and genome-wide binding site analysis, we demonstrate that PafBC (proteasome accessory factor B and C), encoded in the Pup-proteasome system (PPS) gene locus, is the transcriptional regulator of the predominant LexA/RecA-independent pathway. Comparison of the resulting PafBC regulon with the DNA damage response of Mycobacterium smegmatis reveals that the majority of induced DNA repair genes are upregulated by PafBC. We further demonstrate that RecA, a member of the PafBC regulon and principal regulator of the SOS response, is degraded by the PPS when DNA damage stress has been overcome. Our results suggest a model for the regulation of the mycobacterial DNA damage response that employs the concerted action of PafBC as master transcriptional activator and the PPS for removal of DNA repair proteins to maintain a temporally controlled stress response

Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.ISSN:2041-172

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.ISSN:2041-172

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

ISSN:0022-2836ISSN:1089-863

Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response