Faculty Bibliography

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal PAN (proteasome-activating nucleotidase) and eukaryotic proteasomal Rpt1-6 ATPases, such as the N-terminal coiled coil domain, the OB (oligosaccharide/oligonucleotide-binding) domain, and the ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle. Thus, it is unclear how Mpa binds to proteasome core particles. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two out of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP-hydrolysis-driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C-termini to facilitate engagement with proteasome core particles. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.

There is no perfect recipe to balance work and life in academic research. Everyone has to find their own optimal balance to derive fulfilment from life and work.

2020 has been one of the craziest and strangest years we have lived through. Now that it's over, it's an opportunity to show gratitude for all the good things.

Good manners make a difference-in science and elsewhere. This includes our social media etiquette as researchers.

Bacteria use gated proteolytic machines for routine protein quality control and regulated responses to environmental conditions. This review discusses recent advances in understanding the structure and regulation of ClpP proteases, nanomachines widely distributed across bacteria, and the bacterial proteasome, a protease found in relatively few species. For both machines, activators confer substrate specificity. We highlight new data from organisms encoding two ClpP isoforms and the central role of activators as platforms for integrating regulatory signals. Because proteolytic systems contribute to survival and virulence of many bacterial pathogens, understanding their forms and functions enables new approaches to design targeted therapeutics.

COVID-19 has caused a "Hunger Games" like run for emergency funding that risks detracting away from other diseases that ravage humanity.

The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress.IMPORTANCEM. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The bacterium Mycobacterium tuberculosis (Mtb) causes tuberculosis and is responsible for more human mortality than any other single pathogen¹. Progression to active disease occurs in only a fraction of infected individuals and is predicted by an elevated type I interferon (IFN) response^2-7. Whether or how IFNs mediate susceptibility to Mtb has been difficult to study due to a lack of suitable mouse models^6-11. Here, we examined B6.Sst1^S congenic mice that carry the 'susceptible' allele of the Sst1 locus that results in exacerbated Mtb disease^12-14. We found that enhanced production of type I IFNs was responsible for the susceptibility of B6.Sst1^S mice to Mtb. Type I IFNs affect the expression of hundreds of genes, several of which have previously been implicated in susceptibility to bacterial infections^6,7,15-18. Nevertheless, we found that heterozygous deficiency in just a single IFN target gene, Il1rn, which encodes interleukin-1 receptor antagonist (IL-1Ra), is sufficient to reverse IFN-driven susceptibility to Mtb in B6.Sst1^S mice. In addition, antibody-mediated neutralization of IL-1Ra provided therapeutic benefit to Mtb-infected B6.Sst1^S mice. Our results illustrate the value of the B6.Sst1^S mouse to model IFN-driven susceptibility to Mtb, and demonstrate that IL-1Ra is an important mediator of type I IFN-driven susceptibility to Mtb infections in vivo.