You are here
Cilia and flagella are small, hairlike protrusions that are found on the surface of a cell body. They can be sense organs, or they can move, beating in a coordinated motion to either move the cell or to move liquids or small solids across the cell surface. A disease known as primary ciliary dyskinesia (PCD) can cause a number of human disorders. One of these is chronic destructive airway disease, where the cilia of the respiratory system are unable to move mucus out of the airways, and male infertility caused by poor movement of sperm flagella.
Professor Mary Porter (Genetics, Cell Biology, and Development) and her research group and collaborators are studying the proteins that regulate the activity of dynein motors in cilia and flagella. They are using software available through MSI to analyze and compare wild-type and mutant strains in the Chlamydomonas, a type of algae that moves using flagella. Results from studying Chlamydomonas can be applied to research in humans.
In research published last year in the journal Nature Genetics, Professor Porter and her colleagues discovered genetic mutations that are involved in PCD pathogenesis (“The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans,” M. Wirschell, H. Obrich, C. Werner, D. Trischler, R. Bower, W.S. Hale, N.T. Loges, P. Pennekamp, S. Lindberg, U. Stenram, B. Carlen, E. Horak, G. Kohler, P. Nurnberg, G. Nurnberg, M.E. Porter, and H. Omran, Nature Genetics 45, 262-268 (2013)). They have identified the DRC1 subunit of the nexin-dynein regulatory complex, and showed that mutations disrupting DRC1 result in defects that can cause cilia to be defective. This is the first direct evidence that mutations in DRC genes cause human disease.
Image description: Diagrams of the DRC1 subunit in Chlamydomonas (top) and its human counterpart CCDC164 (bottom). The coiled-coil motifs are shown in dark gray. The positions of the protein alterations identified in algae and human are indicated with arrows. (Image and description from M. Wirschell et al., Nature Genetics 45, 262-268 (2013). © Nature Genetics)
posted on March 5, 2014
Bengt Svensson is a Research Associate in the group led by Professor David Thomas (MSI Fellow; Biochemistry, Molecular Biology, and Biophysics). Dr. Thomas specializes in muscle research. Dr. Svensson came to the University as a post-doc in 1999. He worked in the labs of Bridget Barry (BMBB) and David Ferguson (Medicinal Chemistry) before joining the Thomas lab (a.k.a. the Minnesota Muscle Lab) nearly six years ago. He has been using MSI’s HPC and laboratory resources while with the Thomas lab, and also used MSI laboratories during his time in the other groups. His poster was selected as a finalist in the poster competition at the 2013 Research Exhibition. The title of the poster was, “Simulations of a Fluorescent Probe Attached to SERCA.” Dr. Svensson sat down with MSI recently to talk about his research and this poster.
MSI: You’ve been doing similar research to this poster for several years now. You did an article for us in our Research Bulletin, back in 2011.
Bengt Svensson: Yes, this is part of the same research project.
MSI: Why don’t you tell me about what you’re doing in this poster?
BS: Researchers in the David Thomas lab do spectroscopy studies on muscle-related proteins. In the research project presented on the poster the protein studied is SERCA, the Sarco/Endoplasmic Reticulum Ca2+-ATPase. SERCA is the protein that regulates the muscle by making the muscle relax after contraction. It does that by pumping calcium ions in to the Sarco/Endoplasmic Reticulum, lowering the calcium concentration around the muscle fibers.
MSI: So what you’re doing is creating models of how this process works.
BS: Yes, because we have data from spectroscopy measurements that can be interpreted in a structural biology context. We can get information about amount of motion, distances, and relative orientation, but we don’t really know what those mean unless we can create an atomic model that’s actually going to match the experimental data. The experimental results come from samples where we have attached artificial probes to these proteins, in this case fluorescence labels. From simulations we intend to find out the conformation and motion of those probes. Because if the probes are moving really fast, then their locations are going to be averaged out to a central location, but if they’re moving really slow, then every single possible conformation, or position, is going to be important when we’re trying to interpret a distance measurement from spectroscopy. We are aiming to determine whether specific measurements match an atomic model or not.
So simply expressed, this research project, as presented on this poster, is about modeling how the fluorescent probe is behaving while it’s attached to the protein.
MSI: On the poster, you have images of the protein molecule.
BS: Those images are based on an X-ray crystal structure of the SERCA protein. We have a collaborator in Canada, Howard Young [a co-author on the poster], who has crystallized this protein with one of our probes on it. From that crystal structure, however we only know the conformation promoted by crystallization conditions, and crystal contacts but that’s not going to be the only conformation under natural physiological conditions. Because atoms, protein side chains, and protein regions are expected to be moving around quite a bit. This crystal structure does provide an extremely useful snapshot, which I use as the starting point for simulations or different kinds of computational analysis.
The next thing we did was to perform time-resolved fluorescence anisotropy experiments. Fluorescent molecules absorb light and then emit light at a longer wave length. Fluorescence anisotropy experiments measure the depolarization of fluorescence and give us informations about probe mobility. It tells us both the rate and the amplitude of the motion of the fluorescent probe. From anisotropy experiments we would, in the ideal case, expect to see an exponential decay starting from the theoretical maximum value. We noticed that, in our experiments, the curves on the plots don’t start at that maximum. That means that the light detected is already de-polarized to some extent before we can detect it using our fluorescence instrument.
MSI: So that’s telling you that the probe is moving around?
BS: It tells me that something has happened in the probe itself. Bond stretches - vibrational reorganization have occurred causing the fluorescent light to be emitted at an angle different from what was absorbed by the probe. We have to find out exactly what that angle is, because that’s going to be important when we compare the experimental results with data obtained from molecular dynamics simulations. An approach was used that incorporate quantum chemistry calculations of the excited state of the fluorescence probe and varying the angle between the absorption and emission in the analysis of the molecular dynamics simulation data. We were able to determine the information needed to quite accurately predict the experimental anisotropy results from the molecular dynamics simulations.
MSI: You’re using molecular dynamics simulations. Do you use the supercomputers at all, or do you use the labs?
BS: For these molecular dynamics simulations, since I just simulate the small region of the protein around the probe, it doesn’t make sense to use the supercomputers. These simulations, using implicit solvent and a small number of atoms, only scale up to two or four cores. I mostly run my simulations on the lab queue system, the BSCL lab queue. I do have a couple of computers in my office that I use if I’m just going to submit one or two jobs, but for this project it’s important to have enough data for statistical analysis. I have to run multiple simulations, because averaging several data sets is needed to get interpretable results. For this project I have run 18 simulations for 160 ns each; and that is quite a lot of computer simulation time.
MSI: Is there anything else we should know about this project?
BS: What is presented on this poster are the initial steps for us to be able to go on to further studies determining important structural conformations of SERCA based on results from fluorescence spectroscopy. To make sure we could simulate the probe attached to the protein accurately, we compared data from simulations with fluorescence anisotropy measurements. We believe that we have got the needed parameters right, and we can now use our simulations to interpret results from other fluorescence experiments, like FRET. FRET (Fluorescence Resonance Energy Transfer) experiments are used to measure distances between two fluorescence probes that are attached to different parts of the protein. We have several of these experiments going on in the Thomas lab already
Current hypotheses proposed from available crystal structures suggest that some distances between different parts of the protein will change when the protein is preforming its function. One of our approaches is to use specific FRET distances to verify whether a proposed structural model matches distances measured in a specific biochemical state. The structural models can give us a steady-state snapshot of one conformation, but a working protein is not just flipping between static states. Proteins are always flexible to some degree, and that protein dynamics is really important for a lot of protein functions.
For example, as shown in our recent publication (Pallikuth et al., 2013), we observed that in the biochemical high calcium state, the FRET was higher than in the low calcium state, indicating a shorter distance between the cytoplasmic protein domains. In each of these biochemical states, there were multiple structural states observed. Similar conclusions were made from all-atom molecular dynamics simulation of SERCA (Espinoza-Fonseca, & Thomas, 2011). We can actually quantify how much protein dynamics is observed in each individual state. By using a combination of simulation and experiments, we have discovered new insights into how SERCA works during its catalysis.
MSI: You anticipated my next question - this is the sort of research that’s expanding our fundamental knowledge. It’s not drug design.
BS: The parts I’m involved in are not directed to any product or any cures. Of course, once we have a better understanding of how things work, that’s going to help us with those things, too.
MSI: I know there are high blood pressure medications, calcium channel blockers - are they based on the same sort of thing, this system?
BS: Not really. Those calcium channel blockers affect a different system, the calcium channels that are part of the neurochemical signaling that causes contraction. There are some drugs that affect the function of SERCA. For example beta blocker and stimulator drugs which affect SERCA by affecting another protein that is responsible for its regulation. I believe that I described the regulation of SERCA activity by the small protein phospholamban in the previous MSI Research Bulletin article. Phospholamban is affected by beta adrenergic stimulation, which causes phospholamban to be phosphorylated, and less inhibitory of SERCA. That means, it makes SERCA better at relaxing the muscle, reducing stress on the heart. The Thomas lab is collaborating with a company called Celladon and with scientists at the Icahn School of Medicine at Mount Sinai (New York) on a project that targets phospholamban for gene therapy for heart failure.
SERCA is regulating calcium levels in non-muscle cells too, and may be of interest for several other medical reasons. For example, inhibition of SERCA will cause endoplasmic reticulum stress and trigger cell death. SERCA inhibitors could be useful in cancer treatment because of this. I don’t think there are any current uses because of the toxicity of the inhibitors on healthy cells. If one could develop a safe localized delivery into cancer cells, it could work.
The Thomas group has had numerous publications in recent years. A sample of publications that used MSI resources are shown below. MSI PIs are shown in bold text.
- “Phosphorylated Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase,” S Pallikuth, D Blackwell, Z Hu, Z Hou, DT Zieman, B Svensson, DD Thomas, SL Robia, Biophysical Journal, 105:1812-1821 (2013)
- “Nucleotide Activation of the Ca-ATPase,” JM Autry, JE Rubin, B Svensson, J Li, DD Thomas, Journal of Biological Chemistry, 286(46): 39070-39082, DOI:10.1074/jbc.M112.404434 (2012)
- “TRAIL-Induced Death Receptor 5 Networks are Highly Organized,” CC Valley, JD Perlmutter, AK Lewis, AR Braun, CB Karim, DD Thomas, JR Brody, J. R., JN Sachs, Journal of Biological Chemistry, 287(25): 21265–21278 (2012)
- “Atomic-level Characterization of the Activation Mechanism of SERCA by Calcium,” LM Espinoza-Fonseca, DD Thomas, PLoS One, 6:e26936 (2011)
- “Large-scale Opening of Utrophin’s Tandem CH Domains Upon Actin Binding, by an Induced-Fit Mechanism,” AY Lin, E Prochniewicz, ZM James, B Svensson, DD Thomas, Proceedings of the National Academy of Sciences of the USA, 108:12729–12733 (2011)
- “A Continuous Fluorescence Displacement Assay for BioA: An Enzyme Involved in Biotin Biosynthesis,” DJ Wilson, C Shi, BP Duckworth, JM Muretta, U Manjunatha, YY Sham, DD Thomas, CC Aldrich, Analytical Biochemistry (2011)
- “Atomic-level characterization of the activation mechanism of SERCA by calcium.” LM, Espinoza-Fonseca, DD Thomas. PLoS One, 6:e26936 (2011).
- “Phosphorylation-Induced Structural Changes in Smooth Muscle Myosin Regulatory Light Chain,” D Kast, LM Espinoza-Fonseca, C Yi, DD Thomas, Proceedings of the National Academy of Sciences of the USA, 107:8207 (2010)
posted on February 19, 2014
Stem rust, caused by a number of varieties of the fungus Puccinia graminis, is a serious disease of wheat and barley. One variety of wheat stem rust, called Ug99, is particularly virulent, causing up to 100% crop losses. This pathogen affects most varieties of wheat that are grown today. Ug99 has spread throughout eastern Africa, and has also been detected in Yemen and Iran. While there are wheat lines that have been identified as resistant, these lines are not generally the high-yielding varieties that are useful for large-scale farming. The further spread of Ug99 could cause a disaster for food production.
Research Molecular Geneticist Nirmala Jayaveeramuthu and Adjunct Assistant Professor Matthew Rouse (Plant Pathology; USDA-ARS Cereal Disease Laboratory) study wheat-rust resistance genes in wheat and barley. Their lab uses MSI resources to map these genes. Should it be possible to identify molecular markers that are linked to resistance genes, plant breeders might be able to use them to develop strains of wheat resistant to Ug99.
In a project funded by the Bill and Melinda Gates Foundation and the US Department of Agriculture, the Rouse lab has been studying the wheat cultivar Gabo 56, which is resistant to Ug99. They have crossed this cultivar with Chinese spring wheat, a susceptible variety. Both Ugg99-susceptible and resistant progeny have resulted from this crossing. The group is now working with the RISS group to implement a novel method to analyze RNA-Seq data and identify cDNA sequences linked to the resistance gene. Using a de novo transcriptome assembly, transcripts with segregated expression are identified and SNPs are identified and used to create markers to finely map the resistance gene.
Image description: Left: Sample of genetic results of crossing susceptible and resistant plant. Right: Wheat “family tree” with genetic information.
posted on February 5, 2014
Among different types of cancers, prostate cancer has the highest incidence rate among men, and the second-highest death rate. In early stages of prostate cancer, radiation and surgical resection represent the standard of care, and often lead to good outcomes. In advanced cases, various forms of androgen depletion therapies can achieve effective control of the disease. This is because prostate cancer is hormone-dependent, wherein androgens (primarily testosterone and its derivates) fuel the growth of prostate cancer cells. Unfortunately, this only buys the patient some time, because androgen depletion eventually stops working and the cancer becomes resistant to treatment.
The research lab of Assistant Professor Scott Dehm (Masonic Cancer Center) has discovered a new molecular cause of therapeutic resistance - it arises due to the rearrangement of the Androgen Receptor in prostate cancer cells, resulting in the loss of the part of AR that binds androgens. The AR is a steroid hormone receptor transcription factor important for normal prostate function, as well as for the growth of prostate cancer.
Researchers in the Dehm lab are studying the AR using molecular, biochemical, cell biology, and genetic approaches. They are working with members of MSI’s RISS group to explore and characterize the rearrangements that occur in the 180kb Androgen Receptor (AR) locus in models of prostate cancer progression and in clinical tissues from prostate cancer patients. MSI has provided extensive CPU cycles and storage to enable the mapping and identification of structural changes in the genome.
This research has resulted in a recent paper that appeared in the Proceedings of the National Academy of Sciences of the USA. The paper, entitled “TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer” (M.D. Nyquist, Y. Li, T.H. Hwang, L.S. Manlove, R.L. Vessella, K.A.T. Silverstein, D.F. Voytas, S.M. Dehm, PNAS, 11(43):17492, DOI:10.1073/pnas.1308587110 (2013)), describes the discovery of AR gene rearrangements in prostate cancer tissues. It also discusses how the group uses genome engineering to classify the gene rearrangements as drivers of resistance. The researchers hope that this knowledge will help with the development of more effective prostate cancer treatment. (Note: M.D. Nyquist, Y. Li, and T.H. Hwang are MSI users in the Dehm group. D.F. Voytas is an MSI Principal Investigator from the Department of Genetics, Cell Biology, and Development. K.A.T. Silverstein is the MSI RISS Operations Manager and Scientific Lead.)
Other recent publications by the Dehm group that used MSI resources include:
- “Androgen Receptor Splice Variants Mediate Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cell Lines,” Y.M. Li, S.C. Chan, L.J. Brand, T.H. Hwang, K.A.T. Silverstein, S.M. Dehm, Cancer Research, 73:483, DOI: 10.1158/0008-5472.CAN-12-3630 (2013)
- “AR Intragenic Deletions Linked to Androgen Receptor Splice Variant Expression and Activity in Models of Prostate Cancer Progression,” Y. Li, T.H. Hwang, L. Oseth, A. Hauge, R.L. Vessella, S.C. Schmechel, B. Hirsch, K.B. Beckman, K.A. Silverstein, S.M. Dehm, Oncogene, 31:4759-67 (2012)
- “Intragenic Rearrangement and Altered RNA Splicing of the Androgen Receptor in a Cell-Based Model of Prostate Cancer Progression,” Y. Li, M. Alsagabi, D. Fan, G.S. Bova, A.H. Tewfik, S.M. Dehm, Cancer Research, 71:2108-17 (2011)
Image Description: (Top): Structure of testosterone (left) and its derivate dihydrotestosterone. (Bottom): Dihydrotestosterone ligand interactions with the androgen receptor ligand-binding domain binding-pocket. Wild-type complex (gold), shown as a top view of the steroidal plane. The ligand and receptor interact mainly through H-bonds (dotted lines) and hydrophobic interactions. [bottom image adapted from Nat Clin Pract Endocrinol Metab, 2006 March; 2(3): 146-159, DOI: 10.1038/ncpendmet0120]
Posted on January 22, 2014.
Above: Random events may tip the fate of living organisms. Master equations can be used to understand these random events.
Biology is full of randomness – as random molecular events take place in organisms, populations evolve. Master probability equations can provide a complete model of this probabilistic behavior in biomolecular networks. These equations purport to govern all possible outcomes, hence the title “master.” Although these equations have enormous potential in biological research, their complexity has meant that solutions had only been found for the simplest molecular interaction networks.
MSI Principal Investigator and Fellow Yiannis Kaznessis and his PhD student Patrick Smadbeck recently published a paper that describes a numerical closure scheme for the master probability equation that governs random molecular events in chemical or biochemical reactions. The solution involves ordinary differential equations that describe the time evolution of probability distribution moments. The solution by Professor Kaznessis and Mr. Smadbeck will allow researchers to mathematically conceptualize a wide range of experimental observations. Their paper was published in the Proceedings of the National Academy of Sciences of the USA (“A Closure Scheme for Chemical Master Equations,” P Smadbeck, Y Kaznessis, PNAS, 110(35):14261, DOI: 10.1073/pnas.1306481110 (2013)).
Professor Kaznessis and his research group use MSI resources in their work to create new technologies that will be able to fight antibiotic-resistant bacteria. Microorganisms called enterococci have evolved over the years to resist almost all antibiotics, with the result that patients can develop serious infections that medical practitioners are unable to treat. The Kaznessis group uses molecular simulations to study biological interactions and functions.
Posted on January 8, 2014.