Heran Darwin Lab - Microbiology
Heran Darwin Ph.D.
Professor, Department of Microbiology
450 East 29th Street
Alexandria Life Sciences Center (East Tower)
Lab Rm. 321, Office Rm. 329
New York, NY 10016
Office: (212) 263-2624
Lab: (212) 263-2626
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)
ICAAC Young Investigator Award (2006)
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 for NO-resistance and 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 (mycobacterial proteasome ATPase), PafA (proteasome accessory factor A), and Dop (deamidase of Pup). The 20S core particle is a barrel shaped chamber with proteolytic activity. Mpa forms hexamers with ATPase activity similar to eukaryotic proteasomal ATPases that cap the bases of 20S core particle proteases. Dop and PafA are required for the conjugation of Pup to proteins to target them to the proteasome. Importantly, mutants defective for proteasomal degradation do not cause lethal infections in mice.
Small protein modifiers like ubiquitin and SUMO are extensively studied in eukaryotes but had not been found in prokaryotes. Pup is the first protein-to-protein post-translational modifier to be identified in prokaryotes. Mtb enzymes involved in "pupylation" share no structural or enzymatic features with the eukaryotic ubiquitin and ubiquitin-like systems, making them attractive targets for drug development.
We have recently also identified an ATP-independent activator of the mycobacterial proteasome called PafE (proteasome accessory factor E). PafE forms rings that stimulate the degradation of unfolded proteins as well as specific substrates in Mtb. Thus there appears to be at least two independent roads to destruction by the mycobacterial proteasome.
Tuberculosis and copper. We identified a copper-responsive regulon that is dependent on proteasome function for expression, 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 mammals, needs to respond to copper. It is an intriguing possibility that vertebrate hosts use copper to battle invading pathogens, and bacteria have developed mechanisms to resist this host defense. Finally, we are working to understand the link between copper resistance and proteasome-dependent degradation.
An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis.
Jastrab JB, Wang T, Murphy JP, Bai L, Hu K, Merkx R, Huang J, Chatterjee C, Ovaa H, Gygi SP, Li H, Darwin KH.
Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):E1763-72. doi: 10.1073/pnas.1423319112. Epub 2015 Mar 23.
Proteasomal Control of Cytokinin Synthesis Protects Mycobacterium tuberculosis against Nitric Oxide.
Samanovic MI, Tu S, Novák O, Iyer LM, McAllister FE, Aravind L, Gygi SP, Hubbard SR, Strnad M, Darwin KH.
Mol Cell. 2015 Mar 19;57(6):984-94. doi: 10.1016/j.molcel.2015.01.024. Epub 2015 Feb 26.
Copper homeostasis in Mycobacterium tuberculosis.
Shi X, Darwin KH.
Metallomics. 2015 Jan 23. [Epub ahead of print]
The Pup-Proteasome System of Mycobacteria.
Bode NJ, Darwin KH.
Microbiol Spectr. 2014;2(5). pii: http://www.asmscience.org/content/journal/cm/10.1128/microbiolspec.MGM2-....
Shi X, Festa RA, loerger TR, Butler-Wu S, Sacchettini JC, Darwin KH, Samanovic MI.
MBio. 2014 Feb 18;r(1). pii: e00876-13. doi:10.1128/mBio.00876-13.
The Pup-Proteasome System of Mycobacterium tuberculosis.
Samanovic MI, Li H, Darwin KH.
Subcell Biochem. 2013;66:267-95.
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.
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.
"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.
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.
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.
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):485-91
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.
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.
Marie Samanovic-Golden, PhD
Mike Pearce, PhD
Ricky Festa, PhD
Susan Butler-Wu, PhD
Daniel Sinsimer, PhD
Francisca Cerda-Maira, PhD
Kristin Burns, PhD
Xiaoshan "Shirely" Shi, PhD
Jordan Jastrab, PhD
2016 Gordon Research Conference on Microbial Toxins & Pathogenicity:
(Pictured: Drs. Kenneth Cadwell, Heran Darwin, Andrew Darwin, and Victor Torres)