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Introduction



Structural Proteomics In Europe (SPINE) is an integrated research project which brings together some of the top European structural biology institutions in an unprecedented collaborative effort to develop new methods and technologies for high-throughput structural biology.


Background and Overview


The sequencing of the human genome has been hailed as an epoch making achievement ushering in as yet unbounded opportunities for advances in human healthcare. At a pragmatic level it is generally accepted that such advances can only occur when the genomic information is complemented by advances in our understanding of protein function. A full functional description of a protein nowadays requires knowledge of its 3D structure, but the rate of determination of genomic sequences has far outstripped that of structure determination.


The aim of SPINE is to facilitate this process by developing high throughput (HTP) tools for protein over-expression and structure solution. This will involve the introduction of novel robotics techniques into several of the Partner laboratories. SPINE will target the structures of a set of human proteins implicated in disease states, in particular cancer, and neurodegenerative diseases, together with proteins from a set of pathogenic viruses and bacteria, including Herpes viruses and Mycobacter tuberculosis.

 

Our objectives are:

  1. Development of technologies permitting high throughput structure determination by X-ray crystallography and NMR not only of prokaryotic but also of eukaryotic proteins and complexes.
  2. use of these technologies for the determination of the atomic structures of 500+ proteins of medical interest chosen from the following target areas:
    • bacterial pathogens (e.g. M. tuberculosis, B. anthracis) with a focus on virulence genes and potential drug targets
    • viral pathogens including a genomic approach to a complex virus (herpesviridae) and its interacting partners in the host cell, as well as a pan-viral targeting of enzymes proven to be suitable targets for chemotherapy (e.g. replicases and proteases)
    • human proteins or complexes involved in fundamental processes and disease with a focus on protein families relevant to cancer and neuro-degenerative disease (for instance kinases, protease, kiesins, nuclear receptors, cell surface molecules)
  3. establishment of a robust, interactive, dynamic and open network of European centres of excellence, integrating national, international and biotechnological efforts, in which high throughput structure determination is closely integrated with complementary functional and biomedically orientated studies so that it can have a real impact on human health.

Structure based biology will be of paramount importance in pharmaceutics and biotechnology for decades ahead. 

 

The expected achievements fall into several different categories:


3D Structures of proteins relevant to human health: 

We expect to produce 50 structures in one year

    1. Bacterial pathogens: M. tuberculosis and C. jejuni.
    2. Viral pathogens: Herpesviridae and viral enzymes.
    3. Human proteins relevant to cancer.
    4. Human proteins relevant to neurodegenerative diseases.


    The development of new technologies:

    Streamlined, automated procedures for HTP cloning and expression in 96 well format.

      1. A prototype mini workstation for nanolitre drop crystallization and imaging.
      2. Design of improved crystallization screens.
      3. Production of reliable crystal recognition software.
      4. HTP liquid handling for NMR.
      5. European standard for crystal sample mounting for HTP.
      6. Automation of sample handling and MAD data collection at ESRF & Hamburg.
      7. European network of LIMS allowing transparent data exchange.


      Networking:

        1. Industrial platform.
        2. Functioning virtual research centre.


        Training and mobility:

          1. Provision of a forum.
          2. Dissemination of the expertise of the partners throughout the European community.
          3. Yearly open Symposia.

        3D Structures of Proteins Relevant to Human Health: A Detailed Exploration


        The 3D structures of proteins are pivotal for understanding their function and interaction within biological systems. 

        Proteins are the molecular machines that drive nearly every process in the body, from catalyzing chemical reactions to facilitating communication between cells. Understanding the 3D architecture of these proteins is essential for deciphering how they contribute to human health and disease. In this context, Spine Europe 3D aims to generate high-resolution structural data on proteins that are crucial for human health, focusing on those implicated in diseases such as cancer, neurodegenerative disorders, and infections caused by bacterial and viral pathogens.


        1. Significance of 3D Protein Structures in Human Health

        To fully understand the biological role of a protein, it is essential to know its three-dimensional (3D) structure. The 3D structure of a protein is directly tied to its function. Small changes in this structure—due to mutations, binding of ligands, or protein misfolding—can result in diseases. For example:

        • Enzyme Activity: Enzymes rely on their 3D structure to bind substrates and catalyze reactions. Changes in enzyme structure can impair metabolic processes and lead to disorders.
        • Protein-Protein Interactions: Many proteins function by interacting with other proteins. For instance, receptors on cell surfaces need to bind to signaling molecules, and many pathogens (bacteria, viruses) manipulate these interactions to infect host cells.
        • Misfolding and Aggregation: Misfolded proteins can aggregate and form toxic clumps, as seen in neurodegenerative diseases like Alzheimer's or Parkinson's, where protein aggregates disrupt brain function.

        Understanding these structures in detail helps researchers:

        • Identify the underlying causes of diseases.
        • Discover potential biomarkers for early diagnosis.
        • Design therapeutic drugs that can either inhibit or enhance the protein function.



        2. Target Areas for Structural Investigation in Spine Europe 3D

        The primary focus of Spine Europe 3D will be on proteins of medical interest, grouped into several key categories that are relevant to both basic biological research and clinical applications.

        Bacterial Pathogens:

        • Mycobacterium tuberculosis (M. tuberculosis): This bacterium is the causative agent of tuberculosis (TB), a leading cause of death globally. Understanding the structures of proteins involved in the bacterium's survival mechanisms (e.g., cell wall biosynthesis, stress response, and immune evasion) is critical for developing new antibiotics. Key proteins of interest include:
          • M. tuberculosis enoyl-ACP reductase (InhA), which is a target for the antibiotic isoniazid.
          • Mycobacterial ATP synthase, a vital protein for bacterial energy production.
        • Campylobacter jejuni: A major cause of bacterial food poisoning, understanding its virulence factors and its mechanisms of infection can aid in developing more effective treatments. Target proteins might include:
          • Flagellin: Key to bacterial motility and virulence.
          • Cytolethal distending toxin (CDT): A protein that plays a role in DNA damage and cell cycle disruption in host cells.

        Viral Pathogens:

        • Herpesviridae: Herpesviruses are responsible for a range of diseases, from cold sores to potentially fatal conditions in immunocompromised individuals. By understanding the structural properties of herpesvirus proteins, including those involved in viral replication, immune evasion, and cell entry, we can uncover new avenues for antiviral drug design. Critical proteins include:
          • Herpes Simplex Virus Type 1 (HSV-1) Thymidine Kinase, a key enzyme in viral DNA synthesis.
          • Herpesvirus proteases, which are essential for processing viral polyproteins into functional units and are prime targets for antiviral therapies.
        • Viral Enzymes: Many viral pathogens, including HIV and influenza, depend on viral enzymes to replicate. For example:
          • HIV protease: This enzyme is crucial for the maturation of viral particles and is a key target for anti-HIV drugs.
          • Influenza RNA polymerase: Essential for the replication of the virus's RNA genome, and an important target for antiviral drugs.

        Human Proteins Relevant to Cancer:

        Cancer is driven by mutations in genes that control cell growth, differentiation, and apoptosis. Many of the proteins involved in these processes are of considerable interest for drug development. Notable protein families include:

        • Kinases: These enzymes add phosphate groups to other proteins, thereby regulating cellular processes such as division and survival. Targeting kinases involved in cancer, such as EGFR (Epidermal Growth Factor Receptor) or BRAF, has been a highly successful strategy in cancer treatment.
        • Tumor Suppressors: Proteins like p53, which prevent uncontrolled cell division, are often mutated in cancers. Restoring normal function to these proteins is a major therapeutic goal.
        • Cell Surface Molecules: Receptors such as HER2 play a critical role in breast cancer development. Targeting these molecules can inhibit tumor growth.

        Human Proteins Relevant to Neurodegenerative Diseases:

        Proteins implicated in neurodegenerative diseases, such as Alzheimer’s, Parkinson's, and Huntington's, often undergo misfolding and aggregation, leading to cell death and cognitive decline. Key proteins to investigate include:

        • Tau: In Alzheimer’s disease, tau proteins become hyperphosphorylated and form tangles inside neurons, disrupting cellular function. Understanding its 3D structure could help in designing drugs that prevent tau aggregation.
        • α-Synuclein: In Parkinson’s disease, this protein forms toxic aggregates known as Lewy bodies. Structural insights could lead to therapies that stabilize its normal conformation.
        • Huntingtin: Mutations in the huntingtin protein are responsible for Huntington’s disease. Determining its structure and interaction partners may help identify therapeutic targets to prevent its aggregation.