DUB inhibitors _or_ why you should you eat your broccoli

Eat your broccoli!

We’re constantly bombarded by advice on which foods to eat or not eat, but skeptics among us often find compelling evidence for a convincing mechanism of how the foods promote health hard to come by – food has many components, and there are many different cells and metabolic pathways in those cells with which those components interact.

phenethyl isothiocyanate (a component of cruciferous vegetables)

phenethyl isothiocyanate (PEITC, a component of cruciferous vegetables)

Consider broccoli. It is well established that cruciferous vegetables have wide-ranging health benefits, apparently reducing cancer risks and lowering inflammation.  One set of phytochemicals responsible for the potent anti-cancer and anti-inflammatory properties are called isothiocyanates or ‘ITCs’.  It is now four decades since the discovery of ITCs, yet a molecular understanding of what ITCs do in a cell has proven elusive.

In a paper published this month in Cancer Research, Brandeis research scientist Ann Lawson, working in Liz Hedstrom’s laboratory, together with graduate students Marcus Long (Biochem) and Rory Coffey (Mol Cell Biol) and scientists from UbiQ and from Boston College, has shown that ITCs block the action of deubiquitinating enzymes (DUBs),  including the tumorigenesis-associated enzymes USP9x and UCH37, at physiologically relevant concentrations and time scales.

DUB inhibition provides a simple, unifying explanation that can account for many of the diverse health effects of ITCs. Understanding of how ITCs work at the molecular level may, one day, lead to new drug therapies for illnesses such as cancer, chronic inflammation, and neurodegenerative diseases.

Are you ready for your broccoli now? Me, I think I’ll have some kale sprouts.

Lawson AP, Long MJ, Coffey RT, Qian Y, Weerapana E, El Oualid F, Hedstrom L. Naturally occurring isothiocyanates exert anticancer effects by inhibiting deubiquitinating enzymes. Cancer Res. 2015

Sprout Grant Winners Announced

Winners of the 2013 Sprout Grant competition held by the Brandeis Office of Technology and Licensing have been announced. Sprout grants support research that is “novel, patentable and [has] commercial potential“, and encourage students to think about new and different ways to apply their basic science for practical good. Each team applying for a grant must be led by a Brandeis student or postdoc (noted in asterisks below), who were responsible for presenting their proposals to the review panel.

Teams that received funding.

  • Marcus Long (*), Ann Lawson, Lior Rozhansky ’15, and Liz Hedstrom: $20,000 to develop novel inhibitors of deubiquitinating enzymes;
  • Michael Heymann (*), Achini Opathalage, Dongshin Kim, and Seth Fraden: $5,500 for its development of CrystalChip;
  • Michael Spellberg (*), Calla Olson, Marissa Donovan, and Mike Marr: $10,000 to develop a tool to purify Calmodulin-tagged recombinant proteins;
  • Julian Eskin (*) and Bruce Goode: $2,000 for work on a rapid and efficient kit to purify actin;
  • Eugene Goncharov ’13 (*), Yuval Galor ’15,  and Alex Bardasu ’15: $2,500 towards development of their iPhone app LineSaver, which collects data on local hotspots and gives users an estimated wait-time for restaurants, clubs and tourist attractions.

You can read more at BrandeisNOW

Mushroom tyrosinase and the HA tag: a new method for protein labeling

In a new paper in ChemBioChem, researchers from the Hedstrom lab describe a novel method for protein labeling that is versatile and selective.  The method involves the modification of HA tags (a short amino acid sequence commonly used as an epitope tag that contains several tyrosines) selectively in a variety of ways using mushroom tyrosinase. This cheap and versatile chemical biological tool can effect HA tag cleavage, aggregation, or functionalization by changing conditions. The method for dye-labeling HA-tagged proteins has been applied in both E coli and mammalian cell lysates.

Long MJ, Hedstrom L. Mushroom Tyrosinase Oxidizes Tyrosine-Rich Sequences to Allow Selective Protein Functionalization. Chembiochem : a European journal of chemical biology. 2012. (DOI: 10.1002/cbic.201100792)

Greasy tags in Nature

A recent News & Views piece from Nature entitled “Chemical biology: Greasy tags for protein removal” talks about the significance of Long, Gollapalli & Hedstrom’s paper on “Inhibitor Mediated Protein Degradation” (Chem Biol. 2012 May 25;19(5):629-37.) in opening new avenues for drug design

Sprout Grants Awarded to Seven Groups

Another Brandeis NOW story covers the results of the 2012 Sprout Grant competition. Of 20 applications received, half were software related, half life sciences and physical science-related, so the groups were judged separately. Thirteen groups were asked to return for a second round of interviews, coaching and presentations to outside panels of industry judges.  Seven groups were awarded grants:

2012 Sprout Grant winners, life and physical sciences:

Radiation detector, Wellenstein, PI $20,000
  • Tuberculosis treatment, therapeutic, Hedstrom, PI $17,000
  • Cold Stage for Light Microscopy, microscope tools, Turrigiano, PI $16,000
  • Conditional gene silencing, research tool, Lau PI, $6,000

2012 Sprout Grant winners, software:

  • Innermost Labs, social network. Sahar Massachi and Adam Hughes, $7,500
  • Digital Learning Analytics, learning analytics, Larusson PI  $6,000
  • Campus Bash, social network, Y. Sebag, and M. Jafferji $6,500

For more information about the projects and the judging process, read the story at Brandeis NOW.

Who is Selma?

A new paper in Angewandte Chemie International Edition from a Brandeis group led by postdoc Iain MacPherson, Professor of Biology Liz Hedstrom and Assistant Professor of Chemistry Isaac Krauss introduces a new technique they dub SELMA, short for “selection with modified aptamers”. Currently available selection methods can identify the few oligonucleotides in a library of 107 random DNAs or RNAs that bind specifically to a target protein (these specific binders are termed aptamers). However, nucleic acids have a very limited repertoire of chemical functionality — SELMA expands this functionality by introducing an alkyne-modified nucleotide that can be coupled to virtually any azide-containing compound using a copper catalyzed azide-alkyne cycloaddition reaction (“click chemistry“).

The Brandeis group used SELMA to create a library of sugar-modified oligonucleotides and selected for glycoclusters that mimic the epitope of 2G12, an antibody that protects against HIV infection by binding to a cluster of high-mannose glycans on the HIV envelope protein gp120. This is the first example of the application of directed evolution to protein-carbohydrate interactions, a particularly difficult class of interactions to mimic with traditional synthetic methods. Protein carbohydrate interactions are involved in wide array of biological processes, including cell-cell signaling, cell migration and developmental programming as well as immune recognition, so this method should prove very useful.

MacPherson, I. S., Temme, J. S., Habeshian, S., Felczak, K., Pankiewicz, K., Hedstrom, L. and Krauss, I. J. (2011), Multivalent Glycocluster Design through Directed Evolution. Angewandte Chemie International Edition, 50: 11238–11242. doi: 10.1002/anie.201105555


A Little Freedom Makes a Big Difference

As enzymes evolve over time, proteins of similar structure acquire small sequence changes and acquire new activities. What are the key changes in an enzyme’s structure or mechanism that allow this to happen? Researchers from the Hedstrom lab, led by former postdoc Gregory Patton, in collaboration with researchers from the Karolinska Institute, investigated this question in the case of two proteins, inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR). The enzymes share similar structural features but carry out different reactions in a cell. Since the two enzymes are in opposing pathways, there could be severe consequences if the enzymes slip and carry out the ‘other’ reaction.

The results, published last month in Nature Cell Biology, argue strongly that the difference is based on the ability of the enzyme to switch between two conformations. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR, including states in which the cofactor (NAD or NADP) is either in an ‘in’ conformation poised for hydride transfer (below, right), or an ‘out’ conformation in which the cofactor is 6 Å from IMP (below, left).

Using mutagenesis along with kinetic experiments, the group demonstrates that the ‘out’ conformation is required for the deamination of GMP. The accessibility of this conformation at the key step in GMPR but not IMPDH seems to determine the two different outcomes — thus, the freedom of the enzyme and cofactor to carry out a conformational change determines the specificity.

An interesting question, looking at the pathways, is whether GMPR can ‘run in reverse’, catalyzing the direct amination of IMP to form GMP (and saving the cell some energy in the process).  Overexpression of GMPR does allow E. coli to survive in the absence of IMPDH and GMPS, demonstrating that GMPR-driven synthesis of GMP can support life.  Indeed, some modern organisms that live in ammonia-rich environments appear to obtain GMP by this strategy.  If life began in an ammonia rich environment as is often proposed, the ancestral purine biosynthetic pathways may have produced GMP via GMPR.

For more details, see the paper:  Patton GC, Stenmark P, Gollapalli DR, Sevastik R, Kursula P, Flodin S, Schuler H, Swales CT, Eklund H, Himo F, Nordlund P, Hedstrom L. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes. Nat Chem Biol. 2011.

Long receives HHMI fellowship to develop new protein degradation strategy

Marcus Long, a 3rd yr PhD student in the Graduate Program in Biochemistry who works in the Hedstrom Lab, has been awarded a Howard Hughes International Student Predoctoral Research Fellowship for 2011-2013. This award, which is open only to students at selected universities, is given to roughly 40 international students in the life sciences per year in the US. The receipt of this award reflects strongly on the quality of research conducted in Brandeis, and particularly the interdisciplinary approach taken by principal investigator, Prof. Liz Hedstrom. Application for the award requires a clear research plan, which in this instance involves a novel protein degradation strategy (called IMPED), which was pioneered by Prof Hedstrom and her laboratory. Marcus will play his part in a collaborative effort  (alongside lab mates Rory Coffey, Devi Gollapali and established Hedstrom Group collaborators) to understand the mechanism and limitations of this new methodology.

Mehmet Fisek (BS/MS ’08), an alumnus of the Marder lab and undergraduate Neuroscience program at Brandeis, was also among the 48 winners named. Mehmet is currently doing graduate research in Rachel Wilson’s lab in the Dept. of Neurobiology at Harvard, working on olfactory neurophysiology in Drosophila.

See also story at Brandeis NOW.

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