Exploiting On-orbit Crystal properties for Structural Studies of Medically and Economically Important Targets (On-Orbit Crystals) - 07.15.14
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The research entitled "Exploiting On-orbit Crystal properties for Structural Studies of Medically and Economically Important Targets (On-Orbit Crystals)" uses the quasi-microgravity environment of space to grow protein crystals of four different proteins. Understanding the structure of these proteins will help researchers understand how they work, which then could lead to new pharmaceuticals that target the protein. The proteins in this investigation are linked to breast cancer, skin cancer, prion disease and oxidative stress, the latter of which is implicated in many forms of cancer and neurological disorders.
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OpNom CASIS PCG GCF-2
Hauptman-Woodward Medical Research Institute, Buffalo, NY, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
ISS Expedition Duration
March 2014 - September 2014
Previous ISS Missions
- Determination of macromolecular structure has driven major discoveries in biology enabling scientists to visualize macromolecules in unprecedented detail, resulting in profound insights into function and mechanism. Crystals are difficult to produce, often fail to diffract, and when they do, their diffraction may not provide the necessary structural detail. Microgravity crystallization (correctly termed reduced acceleration crystallization) produces crystals of larger volume with improved long-range order when compared to Earth-grown crystals. This is a result of minimized convective surface flows, growth dominated by diffusion, and the crystals remaining suspended in solution for longer periods of time during growth. The improvement in crystal order and volume can be exploited for structural studies. Recent technological advancements including microfocus synchrotron X-ray beams and rapid, shutterless detectors can maximize the signal-to-noise (improving the resolution of the diffraction data) and by careful experimental design allow the capture of structural information close to physiological temperature - shown to be of key importance for an accurate biological understanding of structural dynamics. On-Orbit Crystals aims to make use of these advancements matched to the improved crystal quality to study a set of four proteins that crystallize, but remain structurally uncharacterized. In each case, structural knowledge will have both a significant impact on human health and validate reduced acceleration crystal growth as a means to provide detailed structural and functional insights.
This investigation aims to grow improved quality crystals on orbit. Presently, those grown on the ground are not of sufficient quality to give the necessary structural data. The resulting structural knowledge obtained from on-orbit growth of better diffracting crystals would have significance in explaining mechanism and providing data to speed rational pharmaceutical design in each of the cases under study. Each protein under study is medically and/or economically important. Structural information from each enables an understanding of mechanism .
Crystallization in the reduced acceleration environment of an orbiting spacecraft has been shown to increase both the physical quality (reducing mosaicity) and volume of macromolecular crystals (Snell and Helliwell, 2005; Snell et al., 1995b). While long-scale order is clearly enhanced, the short scale order (key to diffraction resolution) is not directly improved. Brownian motion (a factor reducing short-scale order) is still pervasive at the atomic level in a reduced acceleration environment. Researchers on this proposal will exploit the reduced mosaicity to enhance signal-to-noise by using a continuous rotation data collection method. They can maximize this by matching the beam geometrical spread to the quality of the crystal. The researchers can use the improved quality of crystals grown on orbit to obtain dynamic information on eukaryotic proteins of importance in cancer, oxidative stress and neurological diseases associated with ageing. They go beyond the simple structural snapshot coupling crystallographic studies with computational and solution techniques to probe dynamics of physiological importance.
Four eukaryotic proteins have been selected, human oxidation resistance 1 (OXR1) a vital protein that controls the sensitivity of neuronal cells to oxidative stress (Oliver et al., 2011), human Ethylmalonyl-CoA decarboxylase (ECHDC1) a new metabolite proofreading enzyme (Linster et al., 2011) linked with breast cancer in certain populations (Gold et al., 2008), human Src activating and signaling molecule (Srcasm) associated with cutaneous squamous cell carcinoma (the second most common form of cancer in the United States) (Zhao et al., 2009) and Heat shock 70 kDa protein 13 (HSPA13) implicated in protein misfolding disorders such as prion disease (Grizenkova et al., 2012). Each has limitations that on orbit growth could potentially overcome and enable structural investigations. The resulting structural knowledge would have significance in explaining mechanism and providing data to speed rational pharmaceutical design.
In this work, the researchers use counter diffusion methods for crystallization (Garcia-Ruiz, 2003) on orbit to minimize the deleterious effect of Marangoni convection, prominent with vapor diffusion methods due to the emergence of surface tension driven flow (Boggon et al., 1998; Chayen et al., 1997). Cryopreservation of crystals ruins the long-range quality (Vahedi-Faridi et al., 2003) and masks important biological information (Fraser et al., 2011) so scientists plan to grow and harvest and study the crystals at physiological temperatures. Radiation damage is a problem (Garman and Nave, 2009) that can be overcome with large crystals and a small beam footprint (>10 micron). A complete data set is then collected to compensate for the damage by changing the area of the crystal sampled. By using a microfocus synchrotron X-ray source, a high-speed shutterless detecter (a Pilatus 6M), and fine phi slicing techniques researchers plan to take full advantage of the enhancement in signal to noise associated with reduced mosaicity (Pflugrath, 1999). Extensive ground controls are planned (using counter diffusion, vapor diffusion and batch methods with and without cryopreservation) and will utilize many of the analysis methods pioneered in previous experiments (Chayen et al., 2010; Snell and Helliwell, 2005). These are used to validate the influence of reduced acceleration on the outcome.
Each protein under study is medically and/or economically important. Structural information from each enables an understanding of mechanism. These mechanisms are important as described:
1. OXR1: Oxidative stress is a common etiological feature of neurological disorders (including amyotrophic lateral sclerosis (ALS) and Parkinson's disease) although the pathways that govern defense against reactive oxygen species (ROS) in neurodegeneration remain unclear. In the United States, 50,000-60,000 new cases of PD are diagnosed each year, adding to the one million people who currently have PD. The Center for Disease control rated complications from Parkinson’s disease as the 14th leading cause of death in the United States. ALS most commonly, the disease strikes people between the ages of 40 and 70, and as many as 30,000 Americans have the disease at any given time. Oliver et al (2011) have shown in mice both and cells that loss of Oxr1 causes cell death and that increasing protein levels can protect against ROS. Our target protein Oxr1 is vital for the protection of neuronal cells against oxidative stress and that induction of Oxr1 may be relevant to neurodegenerative pathways in disease. Enhancement of Oxr1 activity in vivo may counteract or even prevent the damage carried out by ROS in the progression of neurodegenerative disorders and its structure and mechanism will provide information key to this development.
2. ECHDC1: A small number of enzymes play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. ECHDC1 is particularly abundant in brown adipose tissue, liver, and kidney in mice and has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity. It may correct a side activity of acetyl-CoA carboxylase and its mutation may be involved in the development of certain forms of ethylmalonic aciduria. Few such “metabolite proofreading enzymes” are known. Mutations in ECHDC1 have been detected in high risk, cancer affected Jewish Ashkenazi women although the nature of the link, if present, is unclear.
3. Srcasm: Src-activating and signaling molecule (Srcasm) downregulates SFK activity and promotes keratinocyte differentiation. Srcasm represents a novel-anti-proliferation gene for keratinocytes (constituting of 95% of the cells in the outermost layer of the skin), and perhaps even a tumor suppressor gene. Srcasm is associated with cutaneous squamous cell carcinoma, the second most common form of nonmelanoma skin cancer and accounts for 20% of cutaneous malignancies (Johnson et al., 1992; Salehi et al., 2007) and 90% of all head and neck cancers.
4. HSPA13: Prion diseases are fatal neurodegenerative disorders that include bovine spongiform encephalopathy (BSE) and scrapie in animals and Creutzfeldt-Jakob disease (CJD) in humans. In 2003 a BSE outbreak in Canada resulted in a loss to the cattle industry exceeding $6 billion (Mitra et al., 2009) – the US cattle industry is 10 times the size of Canada. Scrapie has an economic impact only a fraction of that $10-$20 million. CJD is rare (approximately 250-300 new cases per year in the US) but it is fatal; no treatment is available. HSPA13 is a member of the Hsp70 family of ATPase heat shock proteins, which have been previously implicated in prion propagation. HSPA13 has recently been found to decrease the incubation time of the disease (Grizenkova et al., 2012). The precise role remains to be established but structural knowledge will provide detail to aid this understanding and potential pharmaceutical prevention or treatment.
The diffraction of X-rays and neutrons through crystallized proteins unveils structurally how those proteins are organized. This technique has led to 23 Nobel Prizes, including determining the structure of DNA. Crystallizing proteins in space has been shown in a number of cases to result in larger, more perfect crystals than can be grown on Earth. This investigation aims to harness these capabilities, as well as improved experimental design and recent instrumentation developments, to study protein crystals in high resolution.
The protein structures being studied in On-Orbit Crystals are all unknown, yet each is associated with costly and potentially deadly diseases. The proteins associated with oxidative stress and important for studies on Parkinson’s disease and amyotrophic lateral sclerosis (ALS). Others are human Ethylmalonyl-CoA decarboxylase (ECHDC1), an enzyme linked with breast cancer in certain populations; human Src activating and signaling molecule (Srcasm), which is associated with cutaneous squamous cell carcinoma, the second-most common form of cancer in the United States; and Heat shock 70 kDa protein 13 (HSPA13), which has been linked to prion disorders such as mad cow disease and Creutzfeldt-Jakob disease. Structural information provides the basic knowledge that can help improve the efficiency of drug designs for these diseases. The information from On-Orbit Crystals may help in the fight against all these diseases, and in the long-term could improve the quality of life for millions of people on Earth.
The experiment is passive and requires no resources or interfaces from the ISS. The payload will remain in the Dragon Capsule for a sortie mission and should not be disturbed by the crew. The experiments are activated during the late loading and a buffer gel is used to separate the sample solutions to delay the onset of crystallization until after the payload has reached microgravity.
Experiment is completely passive and requires no on-orbit procedures. The payload should remain in the launch location until return and post mission de-integration.
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