Periodic Reporting for period 4 - HYPROTIN (Hyperpolarized Nuclear Magnetic Resonance Spectroscopy for Time-Resolved Monitoring of Interactions of Intrinsically Disordered Breast-Cancer Proteins)
Reporting period: 2023-09-01 to 2024-08-31
The HYPROTIN project addressed a significant gap in our understanding of tumorigenesis caused by mutations in the BRCA1 gene, particularly related to hereditary breast and ovarian cancer (HBOC). BRCA1 is an intrinsically disordered protein (IDP) that regulates critical cellular processes such as DNA replication. Mutations in BRCA1 disrupt these processes, leading to uncontrolled tissue growth and cancer. The lack of detailed knowledge about the molecular mechanisms of BRCA1’s interactions with other proteins and DNA—its "dark interactome"—limits the development of effective, targeted treatments. Current experimental techniques, such as conventional nuclear magnetic resonance (NMR), are unable to study these weak and transient interactions at the necessary temporal and spatial resolution under physiological conditions.
Breast cancer is one of the most prevalent cancers worldwide, affecting 12% of all women and 0.1% of all men during their lifetimes. For individuals with BRCA1 mutations, the risk increases to as much as 55-65%. Despite the immense social and economic burden of breast and ovarian cancer, mastectomy remains the most common preventive measure due to the absence of precise, targeted therapeutic options. This highlights an urgent need to improve our understanding of the underlying molecular biology to develop more effective, non-invasive treatments. Advancing knowledge of BRCA1 interactions could lead to breakthroughs in drug discovery and early-stage cancer detection, improving patient outcomes and reducing healthcare costs.
The HYPROTIN project had three primary objectives:
1. Methodological Innovation:
- Develop advanced tools for hyperpolarized nuclear magnetic resonance (NMR) to enable atomic-level resolution of BRCA1’s interactions under physiological conditions.
- Establish time-resolved monitoring techniques to study the kinetics of BRCA1’s interactions with other molecules in real-time.
2. Scientific Discovery:
- Decipher the "dark interactome" of BRCA1, focusing on its regulatory network, including interactions with transcription factors (e.g. MYC, MAX) and DNA binding sites (e.g. EBOX).
- Identify the structural and kinetic mechanisms driving these interactions to understand their role in tumorigenesis.
3. Application and Impact:
- Use insights from the interactome to facilitate the development of targeted therapeutic strategies that restore healthy cell cycle regulation in HBOC patients.
- Build a platform for broader applications of hyperpolarized NMR to study other intrinsically disordered proteins, which are implicated in various diseases beyond cancer.
Breast cancer is one of the most prevalent cancers worldwide, affecting 12% of all women and 0.1% of all men during their lifetimes. For individuals with BRCA1 mutations, the risk increases to as much as 55-65%. Despite the immense social and economic burden of breast and ovarian cancer, mastectomy remains the most common preventive measure due to the absence of precise, targeted therapeutic options. This highlights an urgent need to improve our understanding of the underlying molecular biology to develop more effective, non-invasive treatments. Advancing knowledge of BRCA1 interactions could lead to breakthroughs in drug discovery and early-stage cancer detection, improving patient outcomes and reducing healthcare costs.
The HYPROTIN project had three primary objectives:
1. Methodological Innovation:
- Develop advanced tools for hyperpolarized nuclear magnetic resonance (NMR) to enable atomic-level resolution of BRCA1’s interactions under physiological conditions.
- Establish time-resolved monitoring techniques to study the kinetics of BRCA1’s interactions with other molecules in real-time.
2. Scientific Discovery:
- Decipher the "dark interactome" of BRCA1, focusing on its regulatory network, including interactions with transcription factors (e.g. MYC, MAX) and DNA binding sites (e.g. EBOX).
- Identify the structural and kinetic mechanisms driving these interactions to understand their role in tumorigenesis.
3. Application and Impact:
- Use insights from the interactome to facilitate the development of targeted therapeutic strategies that restore healthy cell cycle regulation in HBOC patients.
- Build a platform for broader applications of hyperpolarized NMR to study other intrinsically disordered proteins, which are implicated in various diseases beyond cancer.
The project features an interdisciplinary approach, ranging from studying the structural dynamics of intrinsically disordered transcription factors to developing advanced instrumentation and nuclear magnetic resonance (NMR) methodologies. During the initial phase, protocols for the preparation and production of all target biomolecules were established and optimized. Concurrently, a prototype dissolution dynamic nuclear polarization (DDNP) system was developed specifically to study these biomolecules. DDNP enhances NMR spectroscopy by enabling the acquisition of spectra with significantly increased signal intensities.
Novel NMR detection schemes were designed and implemented to investigate the targets using DDNP. These schemes focused on acquiring high-resolution spectra within the limited lifetime of the signal enhancement provided by DDNP, addressing a critical requirement for the effective use of the technique. Specifically, the following work was performed:
Hyperpolarization-Enhanced NMR Spectroscopy:
-Developed hyperpolarized water-based techniques enabling residue-resolved NMR spectroscopy of proteins at micromolar concentrations, achieving real-time analysis of structural dynamics under near-physiological conditions.
-Investigated mechanisms of hyperpolarization transfer in biomolecular systems, integrating molecular dynamics simulations to clarify the pathways from hyperpolarized water to target biomolecules.
Self-Assembling Peptides for Biomaterials:
-Advanced the design of self-assembling peptides, enabling precise control over silica nanoparticle morphology. This work highlighted the influence of peptide sequence and self-assembly dynamics on the resulting materials, supporting applications in drug delivery and biosensing.
Structural Insights into Protein Interactions:
-Achieved novel residue-specific insights into protein dynamics and their interaction mechanisms under hyperpolarized conditions, enhancing understanding of molecular recognition in key biological systems involving MAX and BRCA1 and its DNA targets.
-Demonstrated the ability to track protein folding, membrane interactions, and exchange processes in real time, offering tools for investigating transient biomolecular phenomena.
Innovative Hyperpolarization Strategies:
-Fremy’s salt was introduced as a low-persistence hyperpolarization agent, combining efficient signal enhancement with rapid radical scavenging to minimize relaxation losses. This advance improves the utility of DNP in a broad range of applications.
-Developed methodologies for selective hyperpolarization transfer to methyl groups in peptides, paving the way for enhanced structural studies of large proteins.
Novel NMR detection schemes were designed and implemented to investigate the targets using DDNP. These schemes focused on acquiring high-resolution spectra within the limited lifetime of the signal enhancement provided by DDNP, addressing a critical requirement for the effective use of the technique. Specifically, the following work was performed:
Hyperpolarization-Enhanced NMR Spectroscopy:
-Developed hyperpolarized water-based techniques enabling residue-resolved NMR spectroscopy of proteins at micromolar concentrations, achieving real-time analysis of structural dynamics under near-physiological conditions.
-Investigated mechanisms of hyperpolarization transfer in biomolecular systems, integrating molecular dynamics simulations to clarify the pathways from hyperpolarized water to target biomolecules.
Self-Assembling Peptides for Biomaterials:
-Advanced the design of self-assembling peptides, enabling precise control over silica nanoparticle morphology. This work highlighted the influence of peptide sequence and self-assembly dynamics on the resulting materials, supporting applications in drug delivery and biosensing.
Structural Insights into Protein Interactions:
-Achieved novel residue-specific insights into protein dynamics and their interaction mechanisms under hyperpolarized conditions, enhancing understanding of molecular recognition in key biological systems involving MAX and BRCA1 and its DNA targets.
-Demonstrated the ability to track protein folding, membrane interactions, and exchange processes in real time, offering tools for investigating transient biomolecular phenomena.
Innovative Hyperpolarization Strategies:
-Fremy’s salt was introduced as a low-persistence hyperpolarization agent, combining efficient signal enhancement with rapid radical scavenging to minimize relaxation losses. This advance improves the utility of DNP in a broad range of applications.
-Developed methodologies for selective hyperpolarization transfer to methyl groups in peptides, paving the way for enhanced structural studies of large proteins.
The project achieved significant advancements across multiple domains, surpassing existing capabilities in nuclear magnetic resonance (NMR) methodologies, biomolecular studies, and biomimetic material design:
Hyperpolarized NMR Spectroscopy:
-Developed hyperpolarized water-based techniques for NMR spectroscopy, enabling the study of proteins at physiological concentrations (micromolar range) with unprecedented sensitivity and temporal resolution. This contrasts with conventional NMR, which requires non-physiological sample concentrations and extended acquisition times.
Real-Time Monitoring of Dynamic Processes:
-Implemented novel NMR detection schemes capable of acquiring high-resolution spectra within the short-lived hyperpolarized states, enabling real-time analysis of fast molecular dynamics and transient biomolecular interactions.
Advanced real-time tracking of solute-to-solid transitions in crystallization processes using hyperpolarized suspensions, offering atomistic insights into mineralization pathways.
Advanced Instrumentation:
Designed and constructed a prototype DDNP system tailored to the investigation of biomolecular systems. This system integrates improved sample transport mechanisms and compatibility with various biomolecules, significantly enhancing the versatility of DDNP.
Combined DDNP with molecular dynamics simulations to elucidate hyperpolarization transfer pathways, advancing the theoretical understanding of magnetization flow in complex systems.
Hyperpolarized NMR Spectroscopy:
-Developed hyperpolarized water-based techniques for NMR spectroscopy, enabling the study of proteins at physiological concentrations (micromolar range) with unprecedented sensitivity and temporal resolution. This contrasts with conventional NMR, which requires non-physiological sample concentrations and extended acquisition times.
Real-Time Monitoring of Dynamic Processes:
-Implemented novel NMR detection schemes capable of acquiring high-resolution spectra within the short-lived hyperpolarized states, enabling real-time analysis of fast molecular dynamics and transient biomolecular interactions.
Advanced real-time tracking of solute-to-solid transitions in crystallization processes using hyperpolarized suspensions, offering atomistic insights into mineralization pathways.
Advanced Instrumentation:
Designed and constructed a prototype DDNP system tailored to the investigation of biomolecular systems. This system integrates improved sample transport mechanisms and compatibility with various biomolecules, significantly enhancing the versatility of DDNP.
Combined DDNP with molecular dynamics simulations to elucidate hyperpolarization transfer pathways, advancing the theoretical understanding of magnetization flow in complex systems.