sign in sign up
<<
Home , Proteomics

EDGE: Today's Science ... Tomorrow's in te Eins e Th : Power to the

everal years ago, three that make life possible: They provide Einstein researchers structural and skeletal support for all organisms, the that regulate Senvisioned a research cellular processes and the compo- project for accomplishing nents of -signaling pathways that, when deranged, can lead to . something novel and poten- The NIAID expressed interest in tially lifesaving: analyze the the Einstein White Paper. Then came 9/11—followed a few months later by proteins of protozoan para- the anthrax-in-the-mail incidents that sites as a way to identify killed five people—and the federal potential targets for drug or focus on biodefense research intensi- fied. In 2003, the NIAID announced vaccine therapy. plans to award five to 10 contracts to create Biodefense Proteomics The researchers—the late George Orr, Research Centers. The key goals of professor of molecular pharmacology; the new research program—“to char- Ruth Hogue Angeletti, professor of acterize of pathogens developmental & molecular and/or host cells [and] to identify and of ; and Louis Weiss, proteins associated with the biology professor of medicine (infectious of the microbes”—closely matched diseases) and —submitted the aims expressed by the Einstein their idea as a white paper early researchers in their white paper. in 2001 to the National Institute of The NIAID listed some 120 organ- Allergy and Infectious Diseases. isms as candidates and grouped Their proposal would involve pro- them into three categories— Photomicroscopist Andrew P. Leonard teomics—the study of the proteins A, B and C (see page 17 for list of took these pictures of the two expressed in a given cell, tissue or selected organisms). Coincidentally, parasites under study at the Einstein organism so as to identify them the Category B organisms included proteomics center. At left are two and determine their structure and Cryptosporidium parvum and C. parvum cysts. At right, invading how they interact with each other. Toxoplasma gondii—the two water- a human fibroblast, is aT. gondii The entire complement of proteins borne intracellular parasites that the tachyzoite (in purple), the life stage expressed in a given cell, tissue or Einstein researchers had proposed that causes disease in humans. Both organism is known as its . for study in their White Paper. And images were taken using a field For the last decade, the spotlight furthermore, Einstein was ideally posi- scanning electron microscope. has shone on —the study tioned for the collaborative effort that of the structure and function of a proteomics center would require. all the genes in an organism. Its “In preparing our proposal for the In 2004, the NIAID announced that centerpiece has been the Human NIAID, it was very easy to put our contracts were being awarded to Project, the effort to map research team together,” recalls Dr. Einstein and six other biodefense pro- the sequence of nucleotides in every Angeletti, the center’s co-principal teomics research centers. Einstein’s single human gene. But this emphasis investigator. “We were in the same contract: an impressive $10.9 million on genes tends to obscure the real building complex—all within a couple for five years. Among all the chosen payoff from studying them. of hundred yards of each other—we proteomics centers, Einstein enjoyed Deciphering —of knew one another and we’d all col- a unique, and noteworthy, distinction. humans and other organisms—is laborated in the past. Plus, Einstein “We are the only proteomics cen- useful mainly for helping scientists has a special strength in infectious ter that does not involve a consortium understand the proteins that genes diseases. So these were our major of several different institutions,” Dr. encode. While genes make proteins strengths in competing for one of Orr noted in the summer of 2004, possible, proteins do all the things those NIAID contracts.” shortly after the contract took effect.

10 Einstein I winter 2008 George Orr Remembered I remember once, while talking about lab problems and how to resolve them, he turned his chair completely around to face me and said, ‘All I ever want is for everybody to be happy.’” Dr. Susan Band Horwitz also has fond memories of Dr. Orr. “George and I published more than 25 papers and reviews together and wrote numer- ous grants with never a harsh word between us,” she recalls. “He was a compassionate and kind human being, he researchers in the Einstein a fine teacher and a superb scientist— T Biodefense Proteomics Research creative, thoughtful and hard working. Center all agree that the center George found science exciting and would not exist without the leader- invigorating, and his enthusiasm was ship of Dr. George Orr, who died in contagious. His willingness to challenge 2005 at age 57. his colleagues with tough questions “George was really the driving made all of us better scientists.” force,” says Ruth Hogue Angeletti, The Einstein Biodefense Proteomics a close collaborator with Dr. Orr in Center “was really the culmination of research and the center’s what George wanted to do and would co-principal investigator. “He had have allowed him to accomplish so the idea for the project, put together many things scientifically,” says Dr. this multidisciplinary team of Einstein Louis Weiss, the proteomic center’s researchers and took the lead in writ- other co-principal investigator. “I have ing up the grant application.” no doubt that George would have “Almost every morning, George developed many new and interesting and I would talk about proteins, technologies tailored specifically to this either in his office or mine,” Dr. biodefense project—and would have Angeletti recalls. “We’d plan experi- had a lot of fun doing so. We’re doing ments, some of which he had thought some of the same sorts of things in his up the previous night while smoking absence. But George—with his great his pipe on his back porch. We would energy, deep thinking, tremendous pro- go over good data from recent days ductivity and careful analysis—is really or try to interpret unexpected results. irreplaceable.” © Andrew Paul Leonard © Andrew

“Instead, all the laboratories involved The two parasites under study at a three-dimensional network of in our contract are located right here Einstein—Cryptosporidium parvum microtubules and filaments known at Einstein. This reflects the fact that and Toxoplasma gondii—belong to as the cytoskeletal scaffold. Located we had all the necessary expertise— the ancient phylum Apicomplexa. beneath the cell’s outer membrane, in terms of biology, proteomics and This phylum contains numerous pro- this dynamic internal membrane —within our own walls.” tozoan pathogens, the most famous structure contains proteins that The Einstein Proteomics Center of which is Plasmodium, the parasite maintain cell shape, anchor internal exemplifies a rapidly growing trend that causes malaria. structures and—thanks to actin and in biomedical research in the United C. parvum and T. gondii have both myosin fibers—gives Apicomplexan States: interdisciplinary programs that been implicated in waterborne dis- organisms their ability to glide. Both marry biology with hard sciences such ease outbreaks caused when people waterborne parasites are discussed in as physics, engineering, mathemat- swallow their oocysts—the resting, more detail in the sidebars on pages ics and computer technology. Such egg-like stage that is highly resistant 13 and 14. programs mark a shift in focus from to chlorination and other water disin- The Einstein researchers won’t individual genes and molecules to a fection techniques. The two parasites attempt to identify the entire “” that encompasses can contaminate water supplies with proteomes of the two parasites, esti- entire organisms and their interacting relative ease, and better treatments mated to number 4,000 proteins in C. networks of genes, proteins, cells and are needed for the infections they parvum and 6,000 in T. gondii. Instead tissues. A key goal of the Einstein pro- cause—the major reasons that both they’re focusing on three “subpro- teomics center, for example, is iden- are considered potential biological teomes” that are thought to contain tifying important protein interactions weapons. proteins crucial to parasite survival that could be interrupted by drugs C. parvum and T. gondii share the and might therefore offer good tar- or vaccines. defining feature of all Apicomplexa: gets for drugs or vaccines:

winter 2008 I Einstein 11 Discovering Protein Interactions arasite proteins that interact that existed among proteins in vivo. combinations for that parasite. Once Pwith other proteins may be good The protein mixture is chopped into the interacting proteins are identified, candidates for targeted therapies. thousands of , some of which a three-dimensional model of the pro- To learn which of the dozens or are cross-linked. Analyzing a cross- tein complex can be created (right). hundreds of proteins in parasite linked with This model shows two homodimeric cells interact to form complexes, yields a spectrum that identifies the (identical) proteins crosslinked at a researchers prepare a mixture of linked peptides (left). To find which pro- contact point (red circle). Proteins that proteins derived from T. gondii or C. teins these linked peptides belong to, turn up in many different protein-pro- parvum cells and add cross-linking researchers map the peptides against a tein interactions are probably critical chemicals to restore connections library consisting of all possible protein to a parasite’s viability.

• The oocyst wall proteins. The Einstein are harvested from the calves’ feces media and broken apart under high proteomics center recently identified and shipped to Einstein. pressure (1,000 pounds per square and characterized all the proteins By contrast, T. gondii is grown in inch). The soluble proteins are then comprising C. parvum’s oocyst wall. tissue culture (human fibroblasts) “fractionated”—separated and in Einstein’s proteomics laboratory. grouped by molecular weight. • Proteins bound to the inner mem- Researchers harvest the tachyzoite Using electron microscopy, brane complex. Lying just beneath stage of the life cycle, which causes researchers examine these fractions, the plasma membrane, this special- disease in humans. or “preps,” to find those containing ized structure contains transport The aims of the Einstein proteomics the three proteomes of interest— proteins, proteins that are crucial for project are to identify parasite pro- oocyst wall, cytoskeleton and internal motility and proteins crucial to the teins (i.e., to correlate experimental membrane. (Below is an electron parasites’ ability to invade host cells. data with the protein predictions micrograph showing a T. gondii prep; derived from the parasites’ genomes) the arrows point to microtubules in • Proteins of the cytoskeletal scaffold. and to characterize them—determine the cytoskeleton.) The proteins are In addition to actin and myosin, the their amino-acid sequences, their further processed in different ways, cytoskeletal scaffold contains tubulin functions, whether they are modified depending on whether they’re proteins and signaling/regulatory after their initial formation (so-called intended for the project’s target complexes including and posttranslational modifications), how validation core or its analytical core. phosphatases. crucial they are to parasite viability, how they compare with proteins in Harvesting and other parasites and humans, and how Fracturing Parasites they interact with each other. (See C. parvum can’t be cultured in vitro, “Protein Interactions” sidebar above.) but its oocysts can be obtained in Before proteins can be identified large amounts from infected ani- and characterized, the parasites must mals. At the Tufts University School be pulverized so their proteins can be of Veterinary Medicine, laboratory- separated. This work is done by the reared, pathogen-free calves are project’s protein separation/develop- infected by a standard strain of C. ment core, headed by Dr. Weiss.

parvum, and the resulting oocysts The parasites are placed in liquid L.M. Weiss Photo courtesy of Dr.

12 Einstein I winter 2008 Asexual Replication

Contamination of food and water

Sexual Oocyst Reproduction Meiosis Life Cycle Illustration: Tatyana Harris Illustration: Tatyana

The Analytical Core Cryptosporidium Parvum Identifying the proteins of these two parasite species begins with the work C. parvum was identified a century ago, but not until 1976 was human of the analytical core, headed by cryptosporidiosis first reported. The parasite lives and replicates in the Dr. Angeletti. An such as intestines of warm-blood animals, which excrete the parasite’s infec- is added to the preps to break tious oocysts in their feces. the proteins into peptides—short When oocysts are swallowed—either from contaminated drinking protein fragments 10 to 20 amino water or food—they rupture, releasing sporozoites that invade intesti- acids long. Then, Dr. Angeletti’s group nal cells. The sporozoites go on to replicate, eventually resulting in the uses mass spectrometry to measure formation of new oocysts. Animals and people infected by swallowing the mass—specifically the mass-to- C. parvum oocysts can experience acute intestinal distress. charge ratio—of the peptides as they Since C. parvum’s tough oocysts are relatively resistant to chlorination, water come off a liquid chromatography treatment that includes proper filtration is crucial for keeping oocysts out of drinking column. (Whole protein molecules water. Waterborne outbreaks attributable to C. parvum are notable for affecting are extremely difficult to analyze in a many thousands of people. Several such outbreaks have been traced to drinking- mass spectrometer, and measuring water sources contaminated by runoff from feedlots containing infected cattle. a whole protein’s mass provides only Acute symptoms of cryptosporidiosis include severe diarrhea, nausea and limited help in identifying it.) abdominal cramps. For most people, these symptoms subside after two or three By revealing the mass of a peptide, weeks. But in people with weakened immune systems such as the elderly or AIDS mass spectrometry helps researchers patients, infection with C. parvum can cause prolonged diarrhea and dehydration determine how many amino acids that can be fatal. comprise that peptide and which The largest known C. parvum outbreak occurred in 1993, when a breakdown in particular amino acids are present. In a Milwaukee water filtration plant resulted in 400,000 cryptosporidiosis cases and addition, mass spectrometry can be contributed to the deaths of more than 50 AIDS and chemotherapy patients. Largely used to reveal the sequence of the to prevent similar C. parvum outbreaks in New York, the city is building a $2.1 bil- amino acids in the peptide. lion filtration plant in Van Cortlandt Park in the Bronx to filter water from the Croton watershed system, which provides New York with 10 percent of its drinking water. The Bioinformatics Core When its genome was sequenced in 2004, C. parvum was described as “a rela- Following mass spectrometry, parasite tively pared-down organism” with only nine million DNA base pairs and eight chro- proteins reduced to tens of thousands mosomes. (By contrast, humans have 3.2 billion base pairs on 23 pairs of chromo- of peptides of known size or mass somes.) Given this paucity of base pairs, it’s not surprising that noncoding, or “junk,” must now be reconstructed with the DNA is virtually absent in C. parvum—less than one percent of the total—making aid of computers—which is where Dr. genomic and proteomic analysis relatively straightforward. However, researchers Andras Fiser comes in. An associate are unable to continuously culture C. parvum in the laboratory, so it’s not susceptible professor in the department of bio- to gene knockouts and other types of genetic manipulation. chemistry at Einstein, Dr. Fiser directs C. parvum’s no-frills genome makes it heavily dependent on its host for nutrition the proteomic center’s bioinformatics and energy—reliance unusual even for a parasite. In contrast to bacteria and most core. Bioinformatics (also called com- other protozoan parasites, for example, C. parvum lacks functioning mitochondria, putational biology) uses computer the energy-producing organelles typically found in cells. technology to analyze the kinds of

winter 2008 I Einstein 13 Contamination of food and water

Acute infection

Oocyst

Tacyzoites Photo courtesy of Dr. L.M. Weiss Photo courtesy of Dr. Above: A host cell nucleus’ view of the invading T. gondii Bradyzoites (tissue cyst) tachyzoite cytoskeleton.

Congenital infection Illustration: Tatyana Harris Illustration: Tatyana

Toxoplasma gondii large data sets generated by genom- ics and proteomics research. T. gondii is one of parasitism‘s great success stories: It can infect Fortunately, the genomes of both T. any warmblooded animal, and it has infected more than half the gondii and C. parvum have recently world’s people, including 50 million Americans. Infected people been sequenced and translated carry thousands of the organisms, many of which reside in the into “ databases,” brain: In contrast to C. parvum, which is content to infect intes- meaning that software programs tinal cells, T. gondii is skilled at penetrating cells throughout the have transformed the long strings of body and crossing the blood-brain barrier. adenines, thymines, guanines and An infection with T. gondii usually feels no worse than a mild cytosines in the parasites’ genomes case of the flu, and the vast majority of infected people experience no serious into genes—and, by extension, into effects. But T. gondii can cause serious brain damage when a healthy immune the amino acid sequences of the system is lacking—in AIDS patients, the elderly, and fetuses, for example. Also, parasites’ proteins. pregnant women who become infected for the first time by T. gondii may pass Dr. Fiser provides the gene predic- it on to their fetuses, who may experience significant brain damage, particu- tion databases to Dr. Angeletti’s larly if infection occurs early in pregnancy. Each year up to 4,000 children in analytical core, which carries out the U.S. are diagnosed with congenital toxoplasmosis. computer-generated (“in silico”) pep- Members of the cat family are largely responsible for T. gondii’s success. All tide predictions on the databases. For cat species can carry T. gondii in their intestines, where the parasite matures example, the enzyme trypsin—often and sexually reproduces to form oocysts. A cat can shed 100 million oocysts in used to break down proteins for mass its droppings after a single infection. spectrometry—always forms peptides Oocyts can survive in the soil for more than a year. They are responsible by cleaving proteins after lysine or for environmentally transmitted T. gondii infections, including: cases in arginine residues. So the analytical which people are exposed to oocysts by handling infected kitty litter; certain core carries out an in silico trypsin foodborne cases of toxoplasmosis (e.g., when people eat salads or other foods digest on the gene prediction data- rinsed in contaminated water); and—perhaps most important—waterborne base to obtain predicted peptides outbreaks caused by T. gondii. Swallowed oocysts release sporozoites, which likely to match the ones obtained by then replicate and ultimately transform into tachyzoites—the life stage that mass spectrometry. rapidly infects organs throughout the body and causes disease in humans. The two groups of data—the The most famous waterborne T. gondii outbreak occurred in Victoria, B.C., experimentally derived peptides and in 1995, when heavy rains overwhelmed a municipal water treatment facility’s the peptides predicted by in silico capacity to filter out oocysts. Some 100 people developed toxoplasmosis, which calculations—are then turned over was traced to contamination from cougar feces. to Dr. Fiser. His task: to “map” the The second route of human infection is through eating undercooked pork or peptides obtained by mass spec- other infected meat. T. gondii forms protective cysts in the muscles and other trometry against the gene prediction tissues of animals (including humans) that it infects. These cysts contain T. databases so parasite proteins can gondii’s bradyozite life stage; when raw or undercooked contaminated meat is be identified and characterized. eaten, bradyozites released in the intestine transform into tachyzoites, which Essentially, Dr. Fiser uses the gene spread and replicate in the body. Toxoplasmosis cases in the U.S. are divided prediction databases to validate equally between those caused by environmental exposure and those from eat- the mass-spectrometry-derived pep- ing infected meat.

14 Einstein I winter 2008 The challenge for the gene prediction programs is identifying the 10 percent of the full genome that codes for “ actual proteins... tides—which, in turn, are used to vali- of previous gene predictions, most the amino acid methionine in all life date the peptides predicted by the involving human and other mam- forms, for example. When scanning databases. It’s an effort that requires malian genomes such as the mouse a DNA sequence that may be thou- repeated cycles—what researchers or monkey. So it’s not surprising that sands of bases long, a gene-predic- call “an iterative process.” The task software “trained” to predict human tion algorithm will ideally select the here is similar to assembling a 10,000- genes and proteins might not do well open reading frame that correctly” piece jigsaw puzzle by constantly on ancient parasites—particularly one determines the amino acid sequence switching your attention between the like T. gondii. encoded by the gene—which is not evolving puzzle and the picture on “This parasite’s 14 chromosomes always easy. As shown in the illustra- the box, but with the added compli- offer a complex genome that we tion below, starting the reading frame cations of initially having many more believe contains about 6,000 genes, at one base instead of another can pieces than are needed to recon- with approximately 88 to 90 percent yield different codons and therefore struct the picture and without seeing of its DNA consisting of non-protein- different amino-acid sequences. all the details of the picture. coding regions,” says Dr. Fiser. “The All these difficulties meant that Dr. Predicting proteins from nucleotide challenge for the gene prediction Fiser and his colleagues would first sequences is not always straight- programs is identifying the 10 per- have to verify the T. gondii gene pre- forward: Many genes can code cent of the full genome that codes dictions before they could validate for more than one protein, and a for actual proteins and to properly T. gondii’s proteins. Their first step gene’s protein product can be modi- reconstruct the splicing of all these was to pool the four gene prediction fied before being translated into a coding pieces, and that’s where they databases for T. gondii to obtain all protein. Fortunately, the effort went make mistakes.” possible genes—a total of 30,000 smoothly with C. parvum and its Failing to recognize T. gondii pro- non-redundant predictions. “Since we uncomplicated genome (see sidebar teins known to be real was just one of expect to find about 6,000 genes inT. on page 13). In fact, the proteomics the problems that Dr. Fiser observed. gondii, this meant that some 24,000 of center recently completed charac- The more common failure of the these gene predictions would turn out terizing the C. parvum oocyst wall gene-prediction software was in pre- to be incorrect,” says Dr. Fiser. proteome. But T. gondii proved a dicting the starting and ending posi- The next step was to take advan- more difficult challenge. tions of proteins—a problem inherent tage of the peptides experimentally “We used four different gene pre- in the nature of the DNA code itself. derived through mass spectrom- diction databases for T. gondii, and a The triplet genetic code is etry and use them to differentiate big surprise for us was the low accu- universal—the DNA nucleotides between real and bogus gene racy of each in predicting proteins adenine-thymidine-guanine code for predictions. This involved mapping —on the order of 30 percent,” says Dr. Fiser. “About 375 clusters of T. gondii proteins had been experimentally derived, and our preliminary results had indicated that these clusters could be used to validate about 1,500 proteins. But our databases rec- ognized only a small fraction of these proteins that we knew were real. This was a sobering experience for all of us—to realize just how far we were from understanding the proteome of this organism even though we had a The sequence of DNA bases above can be read in six possible reading full genomic sequence in hand.” frames—three in the forward (5' to 3') and three in the reverse (3' to 5') direc- One reason for the difficulty stems tion. This illustration of the three forward-direction reading frames shows from the very nature of gene predic- that the same sequence of bases can be interpreted as three entirely different tion databases: They are theoretical, amino-acid sequences depending on whether the gene-prediction algorithm the result of software calculations starts translating at a, t or g (stop codons=*). using algorithms refined by thousands (Diagram courtesy of University of Wisconsin–La Crosse.)

winter 2008 I Einstein 15 the peptides (which were known to Targeting Proteins looking at full genome microarray be “real”) against the gene predic- Then comes the task of winnowing expression patterns,” says Dr. Fiser. tions and looking for “hits”—instances down all those proteins (2,600 have “By combining our protein interaction where experimentally derived pep- now been identified inT. gondii) to and data, we can tides matched up with sections of the achieve the ultimate goal of the pinpoint proteins that are membrane- gene prediction database. Einstein biodefense proteomics bound, experimentally validated by The more extensively a predicted project: find those proteins that mass spectrometry, highly interacting gene was covered by hits, the higher might make good drug targets. For and strongly expressed. Targeting the likelihood that the gene predic- both parasites, that task begins with these proteins may allow us to disrupt tion was valid. “We need to see : matching all essential protein complexes and decent coverage—perhaps 10 or newly identified proteins against the selectively kill the parasite.” 15 percent of the proteins covered human genome—and eliminating by peptide hits that are not overlap- any that resemble human proteins. The Target Validation Core ping—to conclude that a particular “To qualify as a potential drug Once proteins of interest have been predicted gene is valid,” says Dr. Fiser. target, a protein will ideally be unique identified through the proteomics “We never expected that we’d to that parasite and should certainly collaboration of Drs. Angeletti and need to validate T. gondii’s gene be very different from any human Fiser, those proteins are further inves- prediction database, and the process proteins,” says Dr. Fiser. That way, if tigated to establish whether they are has been extremely time-consuming,” a drug is developed that targets the indeed good therapeutic targets. Dr. Fiser notes. “But it represents one protein, it will affect only the parasite These studies are carried out by Dr. of the major achievements of this and not its human host. An extra Weiss, the co-principal investigator of project. This is by far the largest-scale benefit would be if the target shows the Einstein biodefense proteomics empirical validation of the predicted a strong similarity to proteins in other project and co-director of its target genome for any organism.” organisms against which a drug has validation core. The core’s other Once a predicted gene has been already been developed.” co-director is Dr. Kami Kim, professor validated, it can in turn be used as a Potential key proteins are then in the departments of medicine and template for the main task at hand: examined to see if they (or the genes microbiology & immunology. annotating, or conclusively validat- encoding them) interact with other “Part of our task is to establish that ing, the T. gondii proteins them- proteins or genes in the parasite. these proteins are located where selves. As the hits on the database “We know, for example that these they’re predicted to be in the para- accumulate, increasing numbers of parasites have an important inner site,” says Dr. Weiss. He does this by T. gondii proteins are validated and membrane structure that they use to injecting a mixture of its peptide com- identified (matched against the gene invade host cells,” says Dr. Fiser. The ponents into rabbits and obtaining prediction database). The illustra- goal here is to identify proteins critical that are then tagged and tion below shows a protein that has to this membrane’s structure. added to the parasite to see where been partially identified by mapping ”We are analyzing data for more they localize. peptides against the gene prediction than one million known protein inter- “These studies show us database. actions and, to help narrow our focus, what proteins belong to specific structures within the parasites,” says MYVHLVQQGEALAATPLLATEAERTETQKRAERSQCRNVQEGAGGESRRTLPFSGRAAGRVGFFAGGNASPASRR Dr. Weiss. “Over time we build up KRQRPGDRGHCRRSREEARHETDKRTAPGFALCGQASSQSHLFSPQLADEATPNEVARRHFKPVLPPVFSSPTGV inventories of proteins belonging to VTVPCNDTDLVNKQDEVNNAPHVLSAQDQDILASLFPNTINTNFCLLAPASGDRQASSEPLRVGVVLSGGQAAGG critical parasite structures such as the HNVICGIFDYVKRVNPASTVFGFLGGPHGVFSHEYVELTEAIIDKYRNMGGFDMIRSGRHKIETDEQKQKSLEIC cytoskeleton or the internal mem- EKLQLNGLVVIGGDDSNTNAAILAEYFKSKGSSTSVCGCPKTIDGDLKNRFVEISFGFDTACKTYCQQIGNLMRN AMTGGNTYHFVRLMGRSASLITLECALQTHPNYTFIGEEVMAKKQSLRQLVEALVDLVEARYAKGKQYGVVLLPE brane complex.” GLIEFIPEVGVLINEINHIVAAGDFEVSKLTPESRSVFEELPESTRRQLLLDRDPHGNVQVAMIHTEKLLMQMTE To more accurately pinpoint a SELQKRGFQGTFLAQSHYLGYEGRSGYPSDFDATYCYGLGNVAGALIQNKVTACMAVLKDMSSSSNPLDWKAAGI protein’s location, an antiserum can PLTKMMNLETRKGKANVPVIKKFLVDIERPLFQAFAQVRDAMRLEDVYQIPGPMQLNTPTPVLPYTLVGAPSTAS be “affinity purified” so that it contains LLSSSSPQSLGHSRLEFEPLLNPLLLQKETAVVAGAAAHPGAEACNAHIQALFPALGAEAKDFFGGACKLQKAQK antibodies specific to a single pep- IKEKCAVGVVLVGPERPGYANVLCGLVQRVALLGGTVKGFKGARGLLTNDCVVIGEKEAAAQRNQPGFVLLGRTE REEAELFTKEGMKQAAATLQAAGVAALVMIGGTTLHAAVLSELLASQRQPIRVVCVEPSGDLGRFPAHGALQLLK tide. “The crude rabbit antiserum with ELTGKDIVVGSPDAKAMCPGISSTFQQLAGCRGLGFDTETKVASEMIANLLTDSNSAAKYFYFCQVSGGLEAECE its mixture of antibodies is poured onto VGLQTHPNVVLSSQQFKTKTLGEIVTFVADAVKARAALKKNFGVAVINENLFALNKELRDLAVEIHLHFLTHPPQ a column containing the peptide of PASGVCLALTADEEAALMAALSPASRELFTSLPVTFQHKLIRDIEVHQFPKAILRFPAHELIAAMVAAVLKKEKD interest,” Dr. Weiss explains. “We then AGTFSGSFSPLCFEFSDSTERAFQKQDGVSSLGRLHLTGQKKRTQRYWKDVGLGFQTPRLAKESKYVDKKCPFTG wash away everything else and elute NVSIRGRVIKGMVISTKMKRAVVIRRNYLHFVPKYSRFEKRHKNVTCHLSPCFEQVKEGDIVTAGQCRPLSKTIR FNVLKVEKNQVFGNSPQLPESA off only those antibodies that have bound to the peptide of interest, The identity and sequence of this T gondii protein’s 1,297 amino acids were leaving us with a peptide-specific obtained from a gene prediction database. The Einstein researchers com- antiserum. We’ve done that for many pared this prediction with experimental results from mass spectrometry. The of the proteins of interest to us.” colors signify the different levels of confidence in the validity of these peptide The photomicrograph on page matches, or “hits.” Red: more than 60% confident; blue: 30% to 60% confident; 17 shows that the antibody made green: less than 30% confident. against the peptide SAG1 has zeroed

16 Einstein I winter 2008 that comprise T. gondii’s cytoskeletal NIAID Category A, B & C scaffolding. Tubulin, of course, is also Priority Pathogens found in human cells, where it forms the spindle fibers that direct the Category A: High priority agents; movement of chromosomes during include organisms that pose a risk to cell division. Pioneering research in national security because they the laboratory of Einstein’s Susan Band • can easily be disseminated or Horwitz has shown that the drug Taxol transmitted person-to-person inhibits cancer-cell division by target- • cause high mortality, with potential ing these tubulin proteins. But T. gondii for major public health impact tubulin appears to be special. “We have found several different • might cause public panic and tubulin proteins that are highly modi- social disruption fied and seem to localize to specific • require special action for public areas of the T. gondii cytoskeleton,” health preparedness Immunofluorescence microscopy of says Dr. Weiss. “They’re very interest- T. gondii tachyzoites performed in ing and probably relate biologically Examples Dr. Weiss’ laboratory. The tachyzo- to the cytoskeleton’s dynamics and Bacillus anthracis (Anthrax) ites were labeled with an antiserum specialization as an organelle. These Clostridium botulinum (Botulism) made against peptides specific to modified tubulins are possibly unique (Plague) SAG1, a tachyzoite surface antigen to the Apicomplexa—I’m pretty sure Variola major (Smallpox) and that localizes to the parasite’s outer we’ll also find them when we look at other pox viruses membrane. C. parvum’s cytoskeleton—and that Ebola virus uniqueness would make them poten- (Ebola hemorrhagic fever) tially good therapeutic targets.” in on the cell membrane of T. gondii. Dr. Weiss notes that attacking tubu- Category B: second-highest priority Such experiments help to confirm the lins is a tried-and-true strategy. “There agents include those that cellular location of specific peptides are lots of successful antiparasitic and proteins. drugs that target tubulins, such as • are moderately easy to disseminate For any protein of interest, the albenazole for treating both Giardia • cause moderate morbidity and ultimate target-validation strategy is and helminths and mebendazole for low mortality to knock out the gene that codes for treating helminths such as pinworm,” • require enhanced disease surveillance it and observe the effect on the para- he says. “So there’s clearly precedent site. “Ideally, we’ll find that knocking for zeroing in on the cytoskeleton in examples out that gene will prove lethal to the general and tubulins in particular as Coxiella burnetii (Q fever) we develop our list of candidates that parasite, and then we know we’ve Rickettsia prowazekii got an excellent therapeutic target,” may be good protein targets.” (Epidemic typhus) says Dr. Weiss. The responsibility of the Einstein 0157:H7 One problem: no technology exists biodefense proteomics center ends (Diarrheagenic E. coli) for knocking out genes in C. parvum, with identifying targets. “After that, mainly because this parasite can’t it’s up to other researchers to take Vibrio cholerae (Cholera) be cultured throughout its entire life our results and design the drugs or West Nile virus cycle. “What we do here is to heter- vaccines that can eliminate these (West Nile virus encephalitis) ologously express C. parvum genes in parasites as threats to human health,” T. gondii and observe the effect,” says says Dr. Weiss. Category C: Third-highest priority Dr. Weiss. Discoveries made by the Einstein agents include emerging pathogens Alternatively, Dr. Weiss can look proteomics center may also prove that could be engineered for mass for homologs. “In other words,” says useful against one of the world’s dissemination in the future because of Dr. Weiss, “if C. parvum has a gene biggest killers. “We’re badly in need • availability we’re interesting in targeting, is there of more effective drugs against • ease of production and dissemination a closely related version of that gene Plasmodium, which is responsible for in T. gondii? If so, we can study the the more than one million malaria • potential for high morbidity and effect of knocking out that gene in T. deaths that occur each year,” notes mortality and major health impact gondii and extrapolate the findings Dr. Angeletti. “Plasmodium belongs to C. parvum.” to the same Apicomplexa family as Examples our two parasites and resembles them Mycobacterium tuberculosis Tallying the Targets very closely both genomically and (Multidrug-resistant tuberculosis) After vetting hundreds of proteins this proteomically. So we’re hopeful that Other Rickettsia (Rickettsial diseases) way, the researchers are particularly therapeutic targets we identify in Nipah virus (Nipah virus encephalitis) excited about tubulin proteins—the T. gondii and C. parvum may help in Yellow fever virus (Yellow fever) building blocks of the microtubules defeating malaria as well.” E Influenza viruses (Flu) Rabies virus (Rabies)

winter 2008 I Einstein 17