I had great time attending the 2011 edition of the America Association for Cancer research satellite meeting “Molecular Targets and Cancer Therapeutics 2011″ held in San-Francisco. I am very pleased with the overall quality of the meeting and the data presented. It was a fantastic opportunity to expand my background, present our research and connect with various individuals for future collaborative work. Several companies have shown great interest in our work especially regarding our current studies on histone methyl transferase and their specific and selective inhibitors. The poster I presented can be downloaded here (PDF). Below, is the abstract submitted to the meeting.

Background: The nuclear receptor binding SET domain (NSD) protein is a family of three histone-lysine N-methyltransferase (HMTase), NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. NSD1 methylates H3K36 and H4K20 and is associated with acute myeloid leukemia, multiple myeloma, and lung cancer. The NSD1-NUP98 translocation plays a significant role in childhood acute myeloid leukemia with NUP98-NSD1 being an active H3K36 methylase. NSD1 is amplified in multiple myeloma, lung cancer, neuroblastomas and glioblastomas. NSD2 methylates H3K36 and is linked to prostate cancer and multiple myeloma. Over expression of NSD2 in myeloma cells leads to aberrantly high levels of H3K36 di-methylation, accompanied by a decrease in H3K27 methylation. NSD2 is found over expressed in fifteen different cancers and is associated with tumor aggressiveness or prognosis in most types of cancers. NSD3 methylates H3K36 and is associated with both lung and breast cancers along with the acute myeloid leukemia. The amplification of either NSD1 or NSD2 triggers the cellular transformation. NSD3 is found amplified in breast cancer cell lines and primary breast carcinomas. Reducing NSDs activity through specific and selective lysine-HMTase inhibitors appears promising to help suppressing cancer growth.

Little is known about the NSD pathways and our understanding of the histone Lysine-HMTase mechanism is partial. The SET domain of NSD1 has specific mechanisms to recognize histone marks unlike other HMTase. The precise catalytic activities of the NSDs are obscure and discrepancies exist hindering progress in understanding their exact biological functions and pathways in cancer pathogenesis. In this study, we explored the in vitro catalytic activities on histone substrates to understand the substrate recognition and to pave the way for the design of selective and specific NSD inhibitors usable in cancer therapies.

Methods: We used both biochemical and computational methods to understand the substrates recognition by the NSDs and to investigate the structural mechanisms happening in the SET domain during the binding of histone tails.

Results: A key regulatory and a recognition mechanism is driven by the flexibility of a loop at the interface of the SET and postSET region who rotates ~45° and translated 7Å opening the SET domain for the binding of the peptide ligand. This regulatory loop acts as a seat belt for the ligand and contributes to the discrimination and the substrate specificity. In vitro, The SET domain of the NSDs favor H3 recognition and are able to methylate a range of substrate. To reconcile with the in vivo activities previously reported on H3K36 and H4K20, we propose a cross-talk mechanism controlling the substrate recognition.

My lab is having a terrific summer so far. I’m not saying that to make a statement or massage my ego but I am happy to see progress. My two Dutch students (Daan and Lennart) made significant contributions to the projects. With Masayo in maternity leave, I was anxious about slowdowns in the lab. Lennart took some of Masayo duties in managing the day to day lab-life. I must say Lennart is doing a great job. As for myself, I’m stuck in front of my computers writing papers and grant proposals. However, I’m always pleased to discuss results, propose futur experiments, and explain projects anytime with my lab members. My office door is always open, and I like to keep it that way.

A photo of Lennart’s crystal photos of PRMT5, a 72Kda human argine-methyltransferase. This is one of the hits we obtained and we are currently optimizing the crystallization conditions.

Delicate poetry and chef-d’œuvre by Ryan J. Woodward.

Thought of You from Ryan J Woodward on Vimeo.

I won the second place at the art of science image contest during the 2011 annual meeting of the Biophysical Society at Baltimore. Thank you all for voting for my image and congratulations to the winner of the contest, Jorge Bernardino!

My image is the hexameric assembly of the Quinolinate phosphoribosyl transferase enzyme (BNA6) from S. cerevisiae. Quinolinic acid phosphoribosyl transferase (QAPRTase, EC 2.4.2.19) is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the  formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD and clearing the neurotoxin quinolinate. The image shows the hexameric surface with electrostatic properties. The structure was solved by APBS and visualized and rendered with VMD 1.8.7.  The electrostatic field lines are displayed and are “emerging” out of the 3 active sites.

From my 2008 paper “Comprehensive x-ray structural studies of the quinolinate phosphoribosyl transferase (BNA6) from Saccharomyces cerevisiae”; E. di Luccio, and D.K. Wilson, (2008) Biochemistry, 47(13); 4039-50. Epub Mar 6, 2008.

Link to Biophysical Society website.

The last 4 months have been really exciting. The work in the lab has been terrific. Thanks to Masayo’s skills, the projects have been really taking off. We have been doing a bunch of cloning and building numerous constructs of various histone methyltransferase (HMTase). In addition, we started working on some putative regulatory partners. One of our goals is to find highly selective and specific HMTase inhibitors, especially for the NSD family. To do so, we are heavily using virtual ligand screening methods and come-up with a short list of molecules to try in the lab. The latest incarnation of AutoDock, AutoDock Vina, have been very useful and efficient to us. Here an animation of the best candidate we found so far, out of over 10,000 molecules of potential interest.

NSD1 SET domain virtual ligand screening

Recently, Jonathan Eisen at UC Davis tweeted a comment made by a structural biologist at the 18th Annual International Meeting on Microbioal Genomics in California. The comment was along this line “if you don’t study purified enzymes in tube it isn’t science”. Although I understand what the structural biologist meant, I was shocked to hear that type of comment in 2010.

Unfortunately, the heated debate between “experimentalists” and bioinformaticians is far from being over. In a nutshell, the biology world can be roughly divided into 3 worlds. The experimentalists doing only wet biology occupy the first category. They trust only what is inside an Eppendorf tube. At the opposite, we found the second category: the bioinformatics world. The bioinformaticians put into equations what the experimentalists found. Then the bioinformaticians try to understand, and model with an ever-increasing accuracy any biological elements. Their work can be a genomic analysis or predicting the 3D structure of a protein among others. The third category of scientist sits in the middle. They believe that using both wet and dry biology is the right combo to better understand everything.

A fringe of hardcore experimentalist does not believe in all the bioinformatics tools and their power to answer some fundamental questions. Of course, that leads to endless debates amongst scientists. *Yawn* The main argument of experimentalists is “A computer program can’t explain or predict a biological phenomena because there are too many unknowns. If you don’t do wet biology/biochemistry in a tube, it isn’t science. ” Well…sure…Last time I checked, there is a descent number of BS analysis coming from experimentalists. Right? Also, there are numbers of wrong analysis using only bioinformatics too. *Facepalm right here*

As for myself, I am a strong advocate of using the best of both worlds. For instance in structural biology, an X-ray structure gives an accurate snapshot of a 3D structure…but that’s it. What about the flexibility during functions? What about the domain motions? How to accurately understand the mechanism of an enzyme? How does the substrate enter the active site? How the product leaves? How does a protein interact with a partner?

There is a huge gap between the world of known structures and the universe of known protein sequences. Structural genomic projects are unable to keep up with newly discovered genes. How to accurately gain access to nearly all of the 3D protein structure of the whole proteome? Those are just few questions among many more.

Bioinformatics offer many great tools to answers all those questions as long as they are properly used and validated. Unfortunately, some scientists don’t agree with that.  Well! This is 2010, people. It is time to embrace the 30+ years of steady developments in both computer science and bioinformatics and to apply them to biology, broadly define. In science, most of the “easy” stuff has been already unravel. What is left? More difficult and complex problems. In order to make significant contributions in Science, one has to cross boundaries with mixed methods approach. Inter-disciplinary is the way to go!

There are many cool things that can be done when mixing wet “classical” biochemistry and computational biology/biophysics. The virtual ligand screening is one of those things that fall into the major cool category. Nowadays computer have plenty of horsepower that can be put into good use to simulate the binding of libraries of small molecules onto an active site of an enzyme for instance. Following the in silico simulations, the *best* molecules are assessed in the lab for their *experimental* binding/inhibitory properties. In the following video, I used AutodockVina to dock a small subset of 943 compounds into the human SETD1 (NSD1): 20 best docking solutions for each compounds = 18860 docking solutions!

After weeks of ordering lab stuffs, chemicals and equipments, the research projects are taking off. Good data starts accumulating. I’m very pleased with that. Thanks to a grant from the national research foundation of Korea, Masayo joined the lab along with a lab assistant and an undergraduate student. I’m thrilled to see the lab alive and moving forward through our projects. Check-out our lab website: http://webbuild.knu.ac.kr/~diluccio/

My life just got 10x better. I have been notified that the National Research Foundation of Korea (NRF) will fund one of my project (I’m the sole PI) for the next 3 years with a descent amount of money too. Now, I can hire a tech for 3 years along with a couple of students to work on that project and study the 3D structures of the NSD proteins (transcription co-activators – TCA) along with trying to understand the inter-domain flexibility of TCA during functions.
I’m now seriously thinking of publishing some of my preliminary data.

Here some of the figures of my grant.

A nerdy post after such a long break. Sorry

Zinc fingers is typically a domain of about 60 amino acids that fold around one or more zinc ions and is found in over 400 eukaryotic proteins, many of which are involved in the regulation of gene expression and in the maintenance of chromatin structure. Zinc fingers typically show a C4HC3 signature (four cysteines, one histidine, three cysteines) with characteristic cysteine spacing and with additional conserved residues, most notably a tryptophan or other aromatic amino acid preceding the final cysteine pair. Studies have suggested a role for zinc fingers as nucleosome interaction determinants. However their functions are still elusive and controversial, as a variety of functions have been suggested, including phosphoinositide binding and E3 ubiquitin ligase activity. In addition to their role as a DNA-binding module,  zinc finger have been shown to mediate protein-protein and protein-lipid interactions as well.

What about the electrostatic surface properties of zinc-finger domains? Here an example with the models of the 4 zinc-fingers of one of the histone methyl-transferase I’m working on. On the figures, blue is positively charged, and red is negative. The large positive (blue) area will bind to the DNA. But, on the other face, there is room for binding to some positively charged partners. Fascinating!

Saturday 21st, I ran the Turkey Trot’09 (10K) in Davis. It was fun as usual and a somewhat easy 10K.  However the weather was not so great: foggy, wet, slippery and cold. But what a great crowd to cheers us up! I just love Davis and I will miss it very much. This year I ran it in 43 minutes, beating my last year time of 48 minutes. I’m happy with that!….do you like my hair?

Since I have some time-off for the next few weeks before staring my new exiting job, I took advantage of today’s great weather to spend some time flying around, just for the heck of flying. I took off KEDU (UC Davis airport called University airport) using runway 17 then I made a 90 right-turn to the west and climb to 4500ft.

The climb to cruising altitude was really smooth, thanks to the temperature inversion. However the downside of a temperature inversion is bad to poor visibility of such air masses. After few minutes I was overflying Winters and decided to head north for a little while. No other aircrafts were around me and the radio was quiet. Perfect!

After some time, I headed back to Davis and started my descent while turning over Winters. I overflew KEDU at 1600ft then descended a bit to 1400ft to stay underneath SMF airspace. After few moments of flying south Davis, I overflew the city to take some photos of downtown Davis. Not easy to fly with one hand and take pictures with the other.

As usual I had a great time. I can’t get enough of that!

Over the last months, I have been monitoring in disbelief the number of “comments-spams ” that hit my website: 4,464 since March 23. Too me, it is way too many for a website that gets an average of 10 legitimate visits per days. I absolutely can’t wrap my mind around the fact that somewhere an army of bots/zombie PC are burning a lot of CPU and bandwidth to vomit a load of nonsense comments on websites…Thankfully, the spam gets caught in powerful filters such as Akismet. So far 99.955% of the comments are junk on my site…It leads to the question: “How much energy can we save if we take down all the spam-servers?, how much bandwidth can we save?

I finally put together a quickly made movie about some of my flights in California. As you may know by now, is that I love flying. But I love even more sharing it with friends. This video is all about the great times I had so far with my friends, flying around just for the heck of flying or for sightseeing from the sky. California is awesome (obviously), but it is even better from high above…

I recently came across this excellent commentary by Brian Matthews on the 5 (five…) papers Chang et al. retracted back in 2006. For those not familiar with X-ray crystallography and the Changs papers withdraw from leading scientific publishers, I give you a bit of explanation. X-ray crystallography is the gold standard in structure determination and it uses a crystal of pure molecular specie(s) shot through an X-ray beam. If the crystal is good, the electron clouds of the atoms diffract the x-ray beam. By recording various diffractions images, one can compute the electron density inside the crystal and trace (build) the molecular specie(s) in it. Sound simple enough? actually no. I’m not talking here about the maths and physics involved and the phasing problem. The essence of  X-ray crystallography is to solve the phase problem leading to having “good” and “reliable” maps of electron density.

What happened to Chang et al., is that they were working with some wrong electron density maps because of a gross error made early-on during the project pipeline. The culprit was an in-house data reduction program that switched critical column of data. Because of this error, they build/trace various proteins with the “wrong” hand.

Brian Matthews commentary is a solid X-ray crystallography 101 lesson. A lot have been said and written about Chang et al. mistake and their consequences. But, Brian Matthews point out that nowadays we are seeing an ever-increasing use of “black-box” procedures for structure determinations. The rapid development of easy to use X-ray crystallography softwares along with massive computing power render the structure determination fairly easy for one with limited X-ray crystallography knowledge. Solving a structure can be fairly straightforward but it can easily become a tricky task. In any cases but especially in tricky cases, one needs to be extremely cautious about the validity of the maps. Brian Matthews gave us a great lesson about the various checking we all should do when dealing with problems encountered by Chang et al.

X-ray crystallography is like anything else, it is an art. It requires experience, failures, learning from failures and constant knowledge update. Like everything else in Science, it is a grave mistake to assume that we master all the whereabouts of a technology/methodology. I guess, Chang et al. learnt it the hard way. However it raises another question: Shall we seek advice from a peer to help solving a problem in case of dealing with a very hot project? All the 5 retracted papers were all hot projects…

The take home message: be über-cautious and don’t take nice looking maps for granted…