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Tag: St. Jude Children

  • Scientists reveal structures of neurotransmitter transporter

    Scientists reveal structures of neurotransmitter transporter

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    Newswise — (Memphis, Tenn – November 1, 2023) Neurons talk to each other using chemical signals called neurotransmitters. Scientists at St. Jude Children’s Research Hospital have drawn on structural biology expertise to determine structures of vesicular monoamine transporter 2 (VMAT2), a key component of neuronal communication. By visualizing VMAT2 in different states, scientists now better understand how it functions and how the different shapes the protein takes influence drug binding — critical information for drug development to treat hyperkinetic (excess movement) disorders such as Tourette syndrome. The work was published today in Nature.    

    How our neurons talk to each other 

    Chemical compounds called monoamines, which include dopamine, serotonin and adrenaline, play a central role in neuronal communication. These molecules affect how the brain works, controlling our emotions, sleep, movement, breathing, circulation and many other functions. Monoamines are neurotransmitters (signaling molecules) produced and released by neurons, but before they can be released, they must first be packaged into vesicles.  

    Vesicles are cellular compartments that store neurotransmitters before they are released at the synapses (the junction through which chemical signals pass from one neuron to another). Think of vesicles as the cargo ships of the neuronal cell — neurochemicals are packed inside them and taken to where they need to go. VMATs are proteins on the membrane of these vesicles that move monoamines into the space within, acting like loading cranes for the cargo ships.  

    “VMATs are transporters that are required for packing these monoamine neurotransmitters into synaptic vesicles,” explained co-corresponding author Chia-Hsueh Lee, Ph.D., St. Jude Department of Structural Biology.   

    Once the VMAT has packed the vesicle with monoamines, the “cargo ship” moves towards the synaptic gap (the space between neurons), where it releases the chemical compounds.  

    The many faces of monoamine transporters 

    There are two types of VMAT: VMAT1 and VMAT2. VMAT1 is more specialized, found only in neuroendocrine cells, whereas VMAT2 is found throughout the neuronal system and has significant clinical relevance.   

    “We knew that VMAT2 is physiologically very important,” Lee said. “This transporter is a target for pharmacologically relevant drugs used in the treatment of hyperkinetic disorders such as chorea and Tourette Syndrome.” 

    Despite their importance, the structure of VMAT2, which would allow researchers to investigate how it works fully, had remained elusive. Lee and his team used cryo-electron microscopy (cryo-EM) to obtain structures of VMAT2 bound to the monoamine serotonin and the drugs tetrabenazine and reserpine, which are used to treat chorea and hypertension, respectively. This was no easy feat.  

    “VMAT2 is a small membrane protein,” explained co-first author Yaxin Dai, PhD., St. Jude Department of Structural Biology. “This makes it a very challenging target for cryo-EM structure determination.”  

    Despite the difficulty and using some clever tricks, the team captured multiple structures of VMAT2 that allowed them to tease out how the protein functions and investigate how exactly those drugs work. “VMAT transporters adopt multiple conformations [shapes] while transporting their substrate. This is called alternating access transport, where the protein is either “outward” or “inward” facing,” explained co-first author Shabareesh Pidathala, Ph.D., St. Jude Department of Structural Biology. “To completely gain mechanistic understanding at an atomic level, we needed to capture multiple conformations of this transporter.”  

    Answering a 40-year-old question 

    The researchers discovered this dynamic mechanism means multiple opportunities for drugs to bind. They confirmed that reserpine and tetrabenazine bind two different conformations of VMAT2. “30 or 40 years of pharmacological research had suggested that these two drugs bind to the transporter in different ways,” said Pidathala, “but nobody knew the atomic details of how this works. Our structures nicely demonstrate that these two drugs stabilize two different conformations of the transporter to block its activity.” 

    The structure of VMAT2 with serotonin bound allowed the researchers to pinpoint specific amino acids that interact with the neurotransmitter and drive transport. “We believe this is a common mechanism that this transporter uses to engage all the monoamines,” said Lee.  

    While this work offers a huge leap forward in understanding monoamine transport, Lee and his team are delving deeper into its mechanism. For example, the intake of monoamines into vesicles is fueled by protons moving in the other direction. “We identified amino acids that are important for this proton-dependent process,” Lee said, “but we still don’t know how exactly protons drive this transport. Determining this mechanism is our future direction, which will help us to fully appreciate how this transporter works.”  

    Authors and funding 

    The study’s other first author is Shuyun Liao of the School of Life Sciences, Peking University. The study’s co-corresponding author is Zhe Zhang of the School of Life Sciences, Peking University. Other authors include Xiao Li and Chi-Lun Chang of St. Jude, and Changkun Long of the School of Life Sciences, Peking University.  

    The study was supported by grants from National Institutes of Health (R01GM143282), the National Key Research and Development Program of China (2021YFA1302300), the National Natural Science Foundation of China (32171201), the SLS-Qidong innovation fund, the Li Ge-Zhao Ning Life Science Youth Research Foundation, the State Key Laboratory of Membrane Biology of China, and ALSAC, the fundraising and awareness organization of St. Jude. 

     

    St. Jude Children’s Research Hospital  

    St. Jude Children’s Research Hospital is leading the way the world understands, treats and cures childhood cancer, sickle cell disease and other life-threatening disorders. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago. St. Jude shares the breakthroughs it makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of treatment and care for even more children. To learn more, visit stjude.org, read St. Jude Progress blog, and follow St. Jude on social media at @stjuderesearch.   

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  • Predicting condensate formation by cancer-associated fusion oncoproteins

    Predicting condensate formation by cancer-associated fusion oncoproteins

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    Newswise — (Memphis, Tenn – September 28, 2023) Many cancers are caused by fusion oncoproteins, molecules that aberrantly form when a rearrangement of DNA results in parts of two different proteins being expressed as one. Several fusion oncoproteins spontaneously form condensates inside cells that promote cancer development. New research by St. Jude Children’s Research Hospital established a method to study this biophysical process in cells, then used that information as a launchpad to predict the behavior of other fusion oncoproteins. The findings, which offer insight into fusion oncoprotein-driven cancers, were published today in Nature Communications. 

    While genes define everything about us, they are not immutable. Genes are made of DNA, which is constantly being read and replicated. Errors can occur, and sometimes a piece of DNA can break and reattach at a different location. This can lead to two previously independent genes being glued together, resulting in a fusion protein. These unnatural proteins retain properties of both original components, which can have disastrous consequences for cells.  

    “Fusion proteins have been shown to be oncogenic drivers in upwards of 15% of human cancers,” said Richard Kriwacki, Ph.D., St. Jude Department of Structural Biology. These fusion oncoproteins can interfere with cellular regulatory pathways involved in cell growth and differentiation, leading to uncontrolled cell division and cancer.  

     

    Secrets in the droplets 

    “We hypothesized that gaining the ability to form condensates could be linked with the oncogenic properties of fusion oncoproteins,” Kriwacki explained. Biomolecular condensates can form through a process called liquid-liquid phase separation, in which biomolecules separate from the surrounding local environment and form their own compartment, akin to oil droplets in water. Condensates have been shown to be very powerful tools for a cell to regulate many different processes. However, when a fusion oncoprotein has the ability to form a condensate, it can wreak havoc in our cells. 

    Kriwacki, along with collaborators set out to uncover how interwoven fusion oncoproteins were with the process of phase separation.   

     

    The code of fusion oncoprotein condensate behavior 

    The researchers initially examined 166 fusion oncoproteins in cells to observe if they phase separate. Then they categorized them, which was no small feat, according to co-first author Hazheen Shirnekhi, Ph.D., St. Jude Department of Structural Biology. 

    “The condensates were all different sizes, different shapes, and located in different areas of the cell,” Shirnekhi said. “It was difficult for any computer program to recognize the condensates in an unbiased manner, so we had to do this manually. It took a lot of time.”  

    This effort revealed that 58% of the fusion oncoproteins examined formed condensates, opening the door to additional insights.  

    “We found that a large number of those fusion oncoproteins that form condensates, especially in the nucleus, had functional features associated with regulation of gene expression,” Kriwacki said. “The cytoplasmic fusion oncoproteins forming condensates had functional features associated with regulation of cell signaling.” These observations suggest that the fusion oncoproteins elicit their oncogenic properties by altering gene regulation or cell signaling pathways through formation of condensates. 

     

    Machine learning reveals scope of phenomenon  

    In addition to those links to cellular functions, patterns began to emerge within the protein sequences of the fusion oncoproteins that form condensates. These patterns involve so-called physicochemical features, such as number of polar amino acids, charged groups or disordered regions.  

    “When we looked at the sequences of the condensate-forming fusion oncoproteins, we noticed features that are distinct from the condensate-negative fusion oncoproteins,” explained co-first author Swarnendu Tripathi, Ph.D., St. Jude Department of Structural Biology. “That motivated us to select 25 non-redundant features and use data science to predict whether a fusion oncoprotein forms condensates or not.” 

    This data science aspect allowed the researchers to use their 166-sample groundwork to train a machine-learning algorithm using those 25 features. The computational model was then applied to predict the condensate-forming behavior of ~3,000 additional fusion oncoproteins associated with different cancer types.   

    The model predicted that upwards of 67% of those additional fusion oncoproteins likely form condensates. The condensate-forming predictions were tested for a subset of fusion oncoproteins. “The model was shown to be 80% accurate in independent testing with fusions not used in the training,” Tripathi noted. 

    This research establishes the foundational framework for determining the mechanisms underlying the oncogenic properties of fusion oncoproteins to enable their targeted inhibition through pharmaceutical agents or alternative approaches. “We’re looking to address the relationship between condensate formation, alteration of gene expression and oncogenesis,” Kriwacki explained. “We’re working with collaborators so that we can address this causality question in as rigorous a way as possible.” As Kriwacki highlighted, “By obtaining a grasp of the underlying mechanisms, we are setting the stage for potential innovative therapeutic approaches against fusion oncoprotein-driven cancers.” 

     

    Authors and funding 

    The study’s other co-first author was Scott Gorman, formerly of St. Jude. The study’s other authors include Bappaditya Chandra, David Baggett, Cheon-Gil Park, Ramiz Somjee, Benjamin Lang, Seyed Mohammad Hadi Hosseini, Brittany Pioso, Ilaria Iacobucci, Qingsong Gao, Michael Edmonson, Stephen Rice, Xin Zhou, John Bollinger, Madan Babu, Charles Mullighan and Jinghui Zhang, of St. Jude; Diana Mitrea and Michael White, formerly of St. Jude, Yongsheng Li and Stephen Yi of the University of Texas at Austin; Daniel McGrail of Cleveland Clinic; Daniel Jarosz of Stanford University School of Medicine; and Nidhi Sahni of the University of Texas MD Anderson Cancer Center and Baylor College of Medicine.  

    The study was supported by grants from the National Institutes of Health (R35 GM137836, R35 GM133658), Komen Foundation grants (CCR19609287, PDF17483544), the National Cancer Institute (P30 CA021765, R35 CA197695, R01 CA246125, U54 CA243124, R01 CA216391, T32 CA236748, K99 CA240689), the National Institute of General Medical Sciences (F32 GM143847), a St. Jude Children’s Research Hospital Chromatin Collaborative award, a Neoma Boadway Fellowship from St. Jude Children’s Research Hospital, the Cancer Prevention and Research Institute of Texas (RR160021, RP220292), a SummerPlus Program Fellowship from Rhodes College and ALSAC, the fundraising and awareness organization of St. Jude. 

     

     

    St. Jude Children’s Research Hospital  

    St. Jude Children’s Research Hospital is leading the way the world understands, treats and cures childhood cancer, sickle cell disease and other life-threatening disorders. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago. St. Jude shares the breakthroughs it makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of treatment and care for even more children. To learn more, visit stjude.org, read St. Jude Progress blog, and follow St. Jude on social media at @stjuderesearch.   

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  • Mutation accessibility fuels influenza evolution

    Mutation accessibility fuels influenza evolution

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    Newswise — (Memphis, Tenn.—July 28, 2023) The influenza (flu) virus is constantly undergoing a process of evolution and adaptation through acquiring new mutations. Scientists at St. Jude Children’s Research Hospital have added a new layer of understanding to explain why and how flu viruses change. The “survival of the accessible” model provides a complementary view to the more widely recognized “survival of the fittest” way of evolving. The work was published today in Science Advances.  

    Viruses undergo a rapid evolutionary flux due to constant genetic mutations. This rapid flux is why people get a flu shot every year, as we need to tackle the latest flu variant that has emerged as the dominant strain. We often see these mutations in the context of traditional evolutionary thinking, where variant fitness determines which mutated virus emerges as a dominant strain in a population. The St. Jude team investigated this theory and defined an alternative evolutionary principle, which they propose is a key driver of evolution, termed “variant accessibility.” 

    The research, led by Alexander Gunnarsson, Ph.D., and M. Madan Babu, Ph.D., St. Jude Department of Structural Biology and Center of Excellence for Data-Driven Discovery, involved creating a model of mutational accessibility to help predict how and why specific mutations emerge in a population during viral evolution.  

     

    The unappreciated role of variant accessibility 

    The genomic alphabet only has four letters representing the nucleotides: (A)denosine, (T)hymine, (G)uanine, and (C)ytosine. Groups of three nucleotides within a protein-coding gene are called a codon. Codons act like a recipe for assembling proteins, encoding for a specific amino acid. Mutations occur when nucleotides are altered, for instance, during replication. This alteration leads to a different amino acid being used to make the protein. But not all mutations are equally likely to emerge, as Babu and Gunnarsson discovered.  

    “The process of genetic replication has inherent biases built in, such as the relative ease of an A to be mutated to a C rather than to a G,” Babu explained. “This means that the pool of mutants with this A-to-C mutation is larger, and surviving variants will predominantly emerge from that particular pool, even though there may be a fitter sequence with an A-to-G mutation.” 

    Using the influenza virus as a case study, Gunnarsson and Babu translated this concept into a mathematical model. Their model enables researchers to predict the path of future evolution based on the accessibility of a mutation. Of particular interest was exploring how specific protein sites can gain or lose the ability to be modified after acquiring a mutation. They then examined how this gain or loss influenced the protein’s function.  

    Phosphorylation is an example of such a modification. It occurs when a phosphate molecule is added to specific amino acids of a protein. In terms of the flu, phosphorylation can help the virus hijack the host molecular pathways for mediating successful infection. Such mutations may have been critical to influenza pandemics of the past, and it is these datasets that Gunnarsson and Babu used to develop their model. 

     

    The importance of jackpot events 

    The model also helped the researchers better understand a long-conceptualized mutation property, the jackpot event. These are mutations that occur by chance early in the growth of a population, leading to a continuous benefit seen throughout the descendants. “The more accessible a genotype is, the more frequent these specific jackpot events are because it’s simply a probabilistic event,” Gunnarsson explained. “If a particular gene is a hundred times more likely to acquire a specific mutation, you’ll see that jackpot event happening proportionately more frequently. These events are important in evolution and are driven primarily by how accessible the variants are.”  

    More accessible mutations are likely to be predominant in a population even though they may not be the fittest mutation. “If the probability of acquiring the fittest mutation is one out of hundreds of trillions,” Gunnarsson said, “the likelihood of it reaching fixation in a population, even if it’s the fittest mutation, is low. When you have multiple instances of jackpot mutations happening, statistically, the prevalence of this variant increases massively, even if it’s less fit compared to another, more fit but less accessible mutant.” 

     

    Furthering our understanding of mutational bias and predicting outcomes in evolving systems 

    The concept of variant accessibility is elegant in its simplicity, but like most things in nature, it is a balance of statistical probabilities. From the mutation event and differences in the probability of certain nucleotide changes to codon redundancy (multiple codons for the same amino acid), it is a delicate balance between components that drives evolutionary pathways.  

    “Furthering our understanding of biochemical mutational biases (e.g., during replication) in viruses can open up new directions and possibilities because it’ll give much better insights into how a virus is likely to evolve,” Babu stated. In fact, the model is being applied to historical data about how the flu virus has changed within the framework of mutational accessibility to predict viral evolution more accurately.  

    The ability to predict viral evolutionary outcomes based on accessibility has piqued the interest of influenza expert Richard Webby, Ph.D., of St. Jude Department of Host-Microbe Interactions and Director of the World Health Organization Collaborating Centre for Studies on the Ecology of Influenza in Animals and Birds. 

    “There are many scenarios in public health where we try and predict the evolutionary path of influenza viruses, including selecting the most appropriate vaccines for future influenza,” Webby said. “The ‘survival of the accessible’ model will empower these predictions and allow us to identify viruses more likely to take on worrying traits more confidently.” 

    This model also applies beyond influenza or even virology and steers further research into mutational biases in different diseases. In cancer, for example, the model can help answer numerous questions about pathology, such as why particular cancer-driving or drug-resistance mutations repeatedly surface.  

    “Our model can be applied to help predict whether a particular type of mutation is likely to emerge as a tumor driver or as a resistant mutation to a specific treatment,” Babu stated. “We hope our work will spur research into characterizing mutational biases driving viral and tumor evolution. If we can quantify and better understand the biochemical processes contributing to mutational bias, that will be invaluable to predict mutational outcomes in evolving genetic systems. The ability to predict outcomes before they happen will allow us to be prepared when they eventually unfold.” 

     

    Authors and funding 

    The study was supported by grants from the Medical Research Council (MC_U105185859) and ALSAC, the fundraising and awareness organization of St. Jude. 

     

     

    St. Jude Children’s Research Hospital  

    St. Jude Children’s Research Hospital is leading the way the world understands, treats and cures childhood cancer, sickle cell disease and other life-threatening disorders. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago. St. Jude shares the breakthroughs it makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of treatment and care for even more children. To learn more, visit stjude.org, read St. Jude Progress blog, and follow St. Jude on social media at @stjuderesearch.   

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  • Epigenetic landscape modulates pioneer transcription factor binding

    Epigenetic landscape modulates pioneer transcription factor binding

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    Newswise — Memphis, Tenn.—May 24, 2023) Like thread tightly wrapped around a spool, DNA is wrapped around histones and packaged into structures called nucleosomes. Scientists at St. Jude Children’s Research Hospital are exploring how a type of transcription factor called a pioneer transcription factor accesses DNA even when it is tightly wound. Their work revealed how the epigenetic landscape influences transcription factor binding. Problems with transcription have been implicated in numerous cancers, so this more detailed understanding of the process may aid in developing future therapeutics. The study was published today in Nature.  

    The nucleosome packaging of DNA can physically block transcription factors that regulate gene expression from accessing their binding sites. Restricting access to DNA is an integral part of how transcription is regulated. However, pioneer transcription factors can bind to their target piece of DNA even within compacted chromatin and are also known to promote the binding of other transcription factors. 

    Among pioneer transcription factors are the so-called Yamanaka factors which include Oct4 and are used to induce pluripotency (the ability to give rise to different cell types). How pioneer transcription factors access tightly wound DNA was unclear. To better understand the process, scientists at St. Jude used cryo-electron microscopy (cryo-EM) and biochemistry to investigate how Oct4 interacts with nucleosomes.  

    “Building on prior work to understand the dynamic behavior of nucleosomes, we wanted to understand how other factors might utilize those dynamic changes to access chromatin,” said corresponding author Mario Halic, Ph.D., St. Jude Department of Structural Biology. “Oct4 did not bind where we anticipated it might — rather than binding inside the nucleosome, we found that it bound a little bit outside.” 

    “One of the main findings is that epigenetic modifications can affect transcription factor binding and cooperativity,” Halic added. “The existing epigenetic state of chromatin can determine how transcription factors will cooperatively bind to chromatin.” 

    The epigenetic impact 

    Results show that the first Oct4 molecule binding “fixes” the nucleosome in a position that increases the exposure of other binding sites, thus promoting the binding of additional transcription factors and explaining transcription factor cooperativity. They also found that Oct4 contacts histones, and these interactions promote chromatin opening and influence cooperativity. Their work also showed that modifications at histone H3K27 affect the positioning of DNA by Oct4. These findings explain how the epigenetic landscape can regulate Oct4 activity to ensure proper cell programming.  

    Notably, the researchers used endogenous human DNA sequences instead of artificial sequences to assemble their nucleosomes. This allowed them to study the dynamic nature of the nucleosome, despite it being more challenging to work with.  

    “In this work, we used real genomic DNA sequences to study transcription factors in the context of where they function,” said first author Kalyan Sinha, Ph.D., St. Jude Department of Structural Biology. “This strategy allowed us to discover that the first binding event of Oct4 positions the nucleosomal DNA in a manner that allows cooperative binding of additional Oct4 molecules to internal sites. In addition, we observed exciting interactions with histone tails and have seen that histone modifications can alter those interactions. Together, these findings provide new insights into the pioneering activity of Oct4.” 

    “Histone modifications affect how DNA is positioned and how transcription factors can bind cooperatively,” Sinha added, “which means in cells, if you have the same DNA sequence, different epigenetic modifications can result in different, combinatorial effects on transcription factor binding.” 

    Authors and funding 

    The study’s other authors are Silvija Bilokapic, Yongming Du and Deepshikha Malik of St. Jude. 

    The study was supported by the National Institutes of Health (1R01GM135599-01 and 1R01GM141694-01) and ALSAC, the fundraising and awareness organization of St. Jude.  

     

      

    St. Jude Children’s Research Hospital 

    St. Jude Children’s Research Hospital is leading the way the world understands, treats and cures childhood cancer, sickle cell disease and other life-threatening disorders. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago. St. Jude shares the breakthroughs it makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of treatment and care for even more children. To learn more, visit stjude.org, read St. Jude Progress blog, and follow St. Jude on social media at @stjuderesearch. 

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