Defining the function of disease variants with CRISPR editing and multimodal single cell sequencing Baglaenko, Yuriy In: 2024. @article{noKey,
title = {Defining the function of disease variants with CRISPR editing and multimodal single cell sequencing},
author = {Baglaenko, Yuriy},
url = {https://www.biorxiv.org/content/10.1101/2024.03.28.587175v1.abstract},
doi = {https://doi.org/10.1101/2024.03.28.587175},
year = {2024},
date = {2024-01-01},
abstract = {Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. |
A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes Visvanathan, Ramya In: 2024. @article{noKey,
title = {A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes},
author = {Visvanathan, Ramya},
url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0299541},
doi = {https://doi.org/10.1371/journal.pone.0299541},
year = {2024},
date = {2024-01-01},
abstract = {The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes. |
Machine learning accelerates pharmacophore-based virtual screening of MAO inhibitors Cieślak, Marcin In: 2024. @article{noKey,
title = {Machine learning accelerates pharmacophore-based virtual screening of MAO inhibitors},
author = {Cieślak, Marcin},
url = {https://www.nature.com/articles/s41598-024-58122-7},
doi = {https://doi.org/10.1038/s41598-024-58122-7},
year = {2024},
date = {2024-01-01},
abstract = {Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase. |
The transcription factor ZNF469 regulates collagen production in liver fibrosis Steinhauser, Sebastian In: 2024. @article{noKey,
title = {The transcription factor ZNF469 regulates collagen production in liver fibrosis},
author = {Steinhauser, Sebastian},
url = {https://www.biorxiv.org/content/10.1101/2024.04.25.591188v1},
doi = {https://doi.org/10.1101/2024.04.25.591188},
year = {2024},
date = {2024-01-01},
abstract = {Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis. |
Dynamic Clinical Assay Pipeline for Detecting a Virus Williams, Jonathan David In: 2024. @article{noKey,
title = {Dynamic Clinical Assay Pipeline for Detecting a Virus},
author = {Williams, Jonathan David},
url = {https://patents.google.com/patent/US20240141447A1/en},
doi = {US20240141447A1},
year = {2024},
date = {2024-01-01},
abstract = {Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject; capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions; ligating an adapter to create a circular molecule; sequencing the circular molecule to obtain sequence reads; generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus; and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject; capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions; ligating an adapter to create a circular molecule; sequencing the circular molecule to obtain sequence reads; generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus; and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample. |
CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts Huertas, Luis Hernandez In: 2024. @article{noKey,
title = {CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts},
author = {Huertas, Luis Hernandez},
url = {https://www.biorxiv.org/content/10.1101/2024.05.22.595167v1.full},
doi = {https://doi.org/10.1101/2024.05.22.595167},
year = {2024},
date = {2024-01-01},
abstract = {The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. |
Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids Tan, Tao In: 2024. @article{noKey,
title = {Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids},
author = {Tan, Tao},
url = {https://www.sciencedirect.com/science/article/pii/S2666166724002557},
doi = {https://doi.org/10.1016/j.xpro.2024.103090},
year = {2024},
date = {2024-01-01},
abstract = {Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research. |
Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation Zukas, Kieran In: 2024. @article{noKey,
title = {Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation},
author = {Zukas, Kieran},
url = {https://www.sciencedirect.com/science/article/pii/S1538783624003209},
doi = {https://doi.org/10.1016/j.jtha.2024.05.028},
year = {2024},
date = {2024-01-01},
abstract = {Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. |
A ‘rich-get-richer’ mechanism drives patchy dynamics and resistance evolution in antibiotic-treated bacteria Emrah, Şimşek, Kim, Kiyeri, You, Lingchong In: 2024. @article{noKey,
title = {A ‘rich-get-richer’ mechanism drives patchy dynamics and resistance evolution in antibiotic-treated bacteria},
author = {Emrah, Şimşek, Kim, Kiyeri, You, Lingchong},
url = {https://www.embopress.org/doi/pdf/10.1038/s44320-024-00046-5},
doi = {https://doi.org/10.1038/s44320-024-00046-5},
year = {2024},
date = {2024-01-01},
abstract = {Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth. |
Insights into Early Phases of Phycocyanin Crystal Formation via SONICC Spectroscopy Pechkova, Eugenia In: 2024. @article{noKey,
title = {Insights into Early Phases of Phycocyanin Crystal Formation via SONICC Spectroscopy},
author = {Pechkova, Eugenia},
url = {https://www.mdpi.com/2073-4352/14/5/395},
doi = {https://doi.org/10.3390/cryst14050395},
year = {2024},
date = {2024-01-01},
abstract = {This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science. |
Small-Angle X‑ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization Larpent, Patrick In: 2024. @article{noKey,
title = {Small-Angle X‑ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization},
author = {Larpent, Patrick},
url = {https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.4c00418},
doi = {https://doi.org/10.1021/acs.molpharmaceut.4c00418},
year = {2024},
date = {2024-01-01},
abstract = {Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation
and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions
are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them
attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is
not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the
suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased
knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their
particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of
understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This
contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray
scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm
crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect
variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the
physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical
stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization
characterization.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation
and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions
are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them
attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is
not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the
suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased
knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their
particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of
understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This
contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray
scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm
crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect
variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the
physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical
stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization
characterization. |
Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features Roger, Magali In: 2024. @article{noKey,
title = {Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features},
author = {Roger, Magali},
url = {https://pubs.rsc.org/en/content/articlehtml/2024/dt/d3dt03372d},
doi = {10.1039/D3DT03372D},
year = {2024},
date = {2024-01-01},
abstract = {Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated. |
Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP F. Peter, Martin In: 2024. @article{noKey,
title = {Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP},
author = {F. Peter, Martin},
url = {https://www.nature.com/articles/s41467-023-44327-3},
doi = {https://doi.org/10.1038/s41467-023-44327-3},
year = {2024},
date = {2024-01-01},
abstract = {The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa. |
Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket Huang, Chia-Ying In: 2024. @article{noKey,
title = {Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket},
author = {Huang, Chia-Ying},
url = {chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://journals.iucr.org/d/issues/2024/02/00/ud5050/ud5050.pdf},
doi = {https://doi.org/10.1107/S2059798324000329},
year = {2024},
date = {2024-01-01},
abstract = {To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. |
Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome G. Ferens, Fraser, C. Taber, Cassandra In: 2024. @article{noKey,
title = {Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome},
author = {G. Ferens, Fraser, C. Taber, Cassandra},
url = {https://www.nature.com/articles/s42003-024-05904-4},
doi = {https://doi.org/10.1038/s42003-024-05904-4},
year = {2024},
date = {2024-01-01},
abstract = {Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome. |
Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories Zhao, Boyang In: 2024. @article{noKey,
title = {Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories},
author = {Zhao, Boyang},
url = {https://www.nature.com/articles/s41467-024-46627-8#Sec15},
doi = {https://doi.org/10.1038/s41467-024-46627-8},
year = {2024},
date = {2024-01-01},
abstract = {The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication. |
7.10 MAG. A Novel Host Monoacylglyceride for In Meso (Lipid Cubic Phase) Crystallization of Membrane Proteins Krawinski, Pawel In: 2024. @article{noKey,
title = {7.10 MAG. A Novel Host Monoacylglyceride for In Meso (Lipid Cubic Phase) Crystallization of Membrane Proteins},
author = {Krawinski, Pawel},
url = {https://pubs.acs.org/doi/full/10.1021/acs.cgd.4c00087},
doi = {https://doi.org/10.1021/acs.cgd.4c00087},
year = {2024},
date = {2024-01-01},
abstract = {A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature–composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG’s chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature–composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG’s chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid. |
Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9 Ares de Araújo, Evandro In: 2024. @article{noKey,
title = {Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9},
author = {Ares de Araújo, Evandro},
url = {https://www.sciencedirect.com/science/article/pii/S0144861723012043},
doi = {https://doi.org/10.1016/j.carbpol.2023.121739},
year = {2024},
date = {2024-01-01},
abstract = {Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs. |
Pathogenic mutations of human phosphorylation sites affect protein–protein interactions Rrustemi, Trendelina In: 2024. @article{noKey,
title = {Pathogenic mutations of human phosphorylation sites affect protein–protein interactions},
author = {Rrustemi, Trendelina},
url = {https://www.nature.com/articles/s41467-024-46794-8},
doi = {https://doi.org/10.1038/s41467-024-46794-8},
year = {2024},
date = {2024-01-01},
abstract = {Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. |
HEIDI: an experiment-management platform enabling high-throughput fragment and compound screening Metz, A. In: 2024. @article{noKey,
title = {HEIDI: an experiment-management platform enabling high-throughput fragment and compound screening},
author = {Metz, A.},
url = {https://journals.iucr.org/d/issues/2024/05/00/nz5016/index.html},
doi = {https://doi.org/10.1107/S2059798324002833},
year = {2024},
date = {2024-01-01},
abstract = {The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system. |
