Heran Darwin Lab - Microbiology
Heran Darwin Ph.D.
Associate Professor, Department of Microbiology
Medical Science Building
, Room 236
550 First Avenue , New York NY 10016
Office: (212) 263-2624
Fax: (212) 263-8276
Mycobacterium tuberculosis, microbial pathogenesis; molecular biology; proteasome, pupylation, Pup, copper, transcriptional regulation, proteolysis.
1992-1999 PhD, University of California, Los Angeles
1999-2001: Washington University, School of Medicine
2003: Instructor of Microbiology and Immunology
Course director (Advanced Topics in Microbial Pathogenesis)
Irma T Hirschl Award Recipient (2010);
The Tuberculosis Proteasome and Pathogenesis
The pathogen and the disease. Tuberculosis is one of the leading causes of death in the world, killing about 2 million people per year. Nearly one-third of the world is infected with Mtb, which is a rod shaped bacterium that persists in phagocytic cells in the lungs of humans. Although healthy individuals usually control Mtb growth, immunosuppression due to a variety of causes can result in increased bacterial replication and the onset of symptoms. Antibiotic therapy is prolonged (6-9 months) and the failure to comply with treatment can lead to the development of multi-drug and extensively drug resistant strains. New drugs to treat tuberculosis are urgently needed, thus researchers are working to identify activities in Mtb that can be targeted.
The task of finding new drugs and drug targets is hindered by the fact that Mtb is dangerous and slow growing, requiring 2-3 weeks to form colonies on solid media. Furthermore, due to the highly infectious nature of the pathogen, all work must be performed in a biosafety level-3 facility. Taken together, Mtb is one of the most significant and challenging organisms to study.
Mycobacterial resistance to host defenses: the bacterial proteasome. Mtb proteasome activity is required virulence in mice, linking protein degradation to pathogenesis. Proteolysis by the Mtb proteasome requires the 20S proteasome core protease, Pup (prokaryotic ubiquitin-like protein), Mpa (Mycobacterium proteasomal ATPase), PafA (proteasome accessory factor A), and Dop (deamidase of Pup). The 20S core is a barrel shaped chamber with proteolytic activity. Mpa forms hexamers with ATPase activity similar to eukaryotic proteasomal ATPases, and is thought to cap the base of the 20S core. Dop and PafA are required for the conjugation of Pup to proteins to target them to the proteasome. Importantly, 20S core, mpa, dop, and pafA mutants cannot cause lethal infections in mice. Although it is clear that proteasome activity is required for the pathogenesis of Mtb, we do not fully understand why.
Bacterial ubiquitin-proteasome system. Small protein modifiers like ubiquitin and SUMO are extensively studied in eukaryotes but had not been found in prokaryotes until relatively recently. Pup is the first protein-to-protein post-translational modifier to be identified in prokaryotes. The Mtb enzymes involved in "pupylation" share little to no structural or enzymatic features with the eukaryotic ubiquitin systems.
M. tuberculosis and copper. We recently identified a copper-responsive regulon that is dependent on proteasome function and is unique to pathogenic Mycobacterium species. RicR (regulated in copper repressor) regulates five promoters that drive the expression of several genes whose functions are largely unknown. We are currently working to characterize the function of several gene products of this regulon. We are also trying to understand why a pathogen like Mtb, which lives exclusively in humans, needs to respond to copper. Data from several labs suggest that vertebrate hosts use copper to battle invading pathogens, and bacteria have developed mechanisms to resist this host defense.
Copper in microbial pathogenesis: meddling with the metal.
Samanovic MI, Ding C, Thiele DJ, Darwin KH.
2012.Cell Host Microbe. 11:106-15.
Synthesis and evaluation of a selective fluorogenic Pup derived assay reagent for Dop, a potential drug target in Mycobacterium tuberculosis.
Merkx, R.K.E. Burns, P. Slobbe,F. El-Oualid, D. El-Atmioui, K.H. Darwin, H. Ovaa.
2012. ChemBioChem. 13:2056-60.
Mycobacterium tuberculosis prokaryotic ubiquitin-like protein (Pup) deconjugating enzyme Dop is an unusual aspartate amidase.
Burns, K.E., F. McAllister, C. Schwerdtfeger, J. Mintseris, F. Cerda-Maira, E.E. Noens, M. Wilmanns, S.R. Hubbard, F. Melandri, H. Ovaa, S.P. Gygi, and K.H. Darwin.
2012. J. Biol. Chem. In press.
Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.
Cerda-Maira, F., F. McAllister, N.J. Bode, K.E. Burns, S.P. Gygi, and K.H. Darwin.
2011. EMBO Reports. 12:863-70.
A novel copper-responsive regulon in Mycobacterium tuberculosis.
Festa RA, Jones MB, Butler-Wu S, Sinsimer D, Gerads R, Bishai WR, Peterson SN, Darwin KH.
Mol Microbiol. 2011 Jan;79(1):133-48. PMID: 21166899
Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation.
Wang T, Darwin KH, Li H.
Nat Struct Mol Biol. 2010 Nov;17(11):1352-7. PMID: 20953180
"Depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.
Burns KE, Cerda-Maira FA, Wang T, Li H, Bishai WR, Darwin KH.
Mol Cell. 2010 Sep 10;39(5):821-7. PMID: 20705495
Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
Cerda-Maira FA, Pearce MJ, Fuortes M, Bishai WR, Hubbard SR, Darwin KH.
Mol Microbiol. 2010 Sep;77(5):1123-35. PMID: 20636328
Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates.
Burns KE, Pearce MJ, Darwin KH.
J Bacteriol. 2010 Jun;192(11):2933-5. PMID: 20233925
Prokaryotic ubiquitin-like protein (Pup) proteome of Mycobacterium tuberculosis [corrected] .
Festa RA, McAllister F, Pearce MJ, Mintseris J, Burns KE, Gygi SP, Darwin KH.
PLoS One. 2010 Jan 6;5(1):e8589.
Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa.
Wang T, Li H, Lin G, Tang C, Li D, Nathan C, Darwin KH, Li H.
Structure. 2009 Oct 14;17(10):1377-85.
Prokaryotic ubiquitin-like protein pup is intrinsically disordered.
Chen X, Solomon WC, Kang Y, Cerda-Maira F, Darwin KH, Walters KJ.
J Mol Biol. 2009 Sep 11;392(1):208-17.
Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis.
Nat Rev Microbiol. 2009 Jul;7(7):485-91PMID:19483713
Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis.
Pearce MJ, Mintseris J, Ferreyra J, Gygi SP, Darwin KH.
Science. 2008 Nov 14;322(5904):1104-7. PMID:18832610
Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis.
Festa RA, Pearce MJ, Darwin KH.
J Bacteriol. 2007 Apr;189(8):3044-50. PMID:17277063
Xiaoshan "Shirely" Shi
Marie Samanovic-Golden, PhD
Mike Pearce, PhD
Ricky Festa, PhD
Susan Butler-Wu, PhD
Daniel Sinsimer, PhD
Francisca Cerda-Maira, PhD
Kristin Burns, PhD