RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories Kaundal, Soni In: 2024. @article{noKey,
title = {RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories},
author = {Kaundal, Soni},
url = {https://www.science.org/doi/full/10.1126/sciadv.adp9333},
doi = {https://doi.org/10.1126/sciadv.adp9333},
year = {2024},
date = {2024-12-20},
abstract = {Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. |
Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening Dunnett, Louise In: 2024. @article{noKey,
title = {Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening},
author = {Dunnett, Louise},
url = {https://www.biorxiv.org/content/10.1101/2024.12.17.628909v1.abstract},
doi = {https://doi.org/10.1101/2024.12.17.628909},
year = {2024},
date = {2024-12-18},
abstract = {The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation. |
Engineering and structures of Crimean-Congo hemorrhagic fever virus glycoprotein complexes McFadden, Elizabeth In: 2024. @article{noKey,
title = {Engineering and structures of Crimean-Congo hemorrhagic fever virus glycoprotein complexes},
author = {McFadden, Elizabeth},
url = {https://www.cell.com/cell/fulltext/S0092-8674(24)01325-4},
doi = {https://doi.org/10.1016/j.cell.2024.11.008},
year = {2024},
date = {2024-12-18},
abstract = {Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus that can cause severe disease in humans with case fatality rates of 10%–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we use structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-electron microscopy (cryo-EM) structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus that can cause severe disease in humans with case fatality rates of 10%–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we use structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-electron microscopy (cryo-EM) structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. |
Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon Thermococcus kodakarensis Naz, Zahra In: 2024. @article{noKey,
title = {Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon Thermococcus kodakarensis},
author = {Naz, Zahra},
url = {https://www.mdpi.com/1422-0067/25/23/12893},
doi = {https://doi.org/10.3390/ijms252312893},
year = {2024},
date = {2024-11-30},
abstract = {Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea. |
Allosteric substrate release by a sialic acid TRAP transporter substrate binding protein Schneberger, Niels In: 2024. @article{noKey,
title = {Allosteric substrate release by a sialic acid TRAP transporter substrate binding protein},
author = {Schneberger, Niels},
url = {https://www.nature.com/articles/s42003-024-07263-6},
doi = {https://doi.org/10.1038/s42003-024-07263-6},
year = {2024},
date = {2024-11-23},
abstract = {The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules. |
Viral sequence determines HLA-E-restricted T cell recognition of hepatitis B surface antigen Murugesan, Gavuthami In: 2024. @article{noKey,
title = {Viral sequence determines HLA-E-restricted T cell recognition of hepatitis B surface antigen},
author = {Murugesan, Gavuthami},
url = {https://www.nature.com/articles/s41467-024-54378-9},
doi = {https://doi.org/10.1038/s41467-024-54378-9},
year = {2024},
date = {2024-11-22},
abstract = {The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules. |
Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3 Chuang, Hsiu-Chun, Ruidong, Li, Li, Li, Kai-Hui, Sun In: 2024. @article{noKey,
title = {Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3},
author = {Chuang, Hsiu-Chun, Ruidong, Li, Li, Li, Kai-Hui, Sun},
url = {https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-024-11036-0},
doi = {https://doi.org/10.1186/s12864-024-11036-0},
year = {2024},
date = {2024-11-21},
abstract = {We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples.},
keywords = {MANTIS},
pubstate = {published},
tppubtype = {article}
}
We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples. |
Engineering of soluble bacteriorhodopsin Nikolaev, Andrey In: 2024. @article{noKey,
title = {Engineering of soluble bacteriorhodopsin},
author = {Nikolaev, Andrey},
url = {https://www.biorxiv.org/content/10.1101/2024.11.20.624543v1.abstract},
doi = {https://doi.org/10.1101/2024.11.20.624543},
year = {2024},
date = {2024-11-21},
abstract = {Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein. Here, we report engineering of soluble analogues of bacteriorhodopsin, NeuroBRs, which bind retinal and photocycle under illumination. We also report the crystallographic structure of NeuroBR_A, determined at anisotropic resolution reaching 1.76 Å, that reveals a conserved chromophore binding pocket and tertiary structure. Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein. Here, we report engineering of soluble analogues of bacteriorhodopsin, NeuroBRs, which bind retinal and photocycle under illumination. We also report the crystallographic structure of NeuroBR_A, determined at anisotropic resolution reaching 1.76 Å, that reveals a conserved chromophore binding pocket and tertiary structure. Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins. |
Broadening alloselectivity of T cell receptors by structure guided engineering Karuppiah, Vijaykumar In: 2024. @article{noKey,
title = {Broadening alloselectivity of T cell receptors by structure guided engineering},
author = {Karuppiah, Vijaykumar},
url = {https://www.nature.com/articles/s41598-024-75140-7#Abs1},
doi = {https://doi.org/10.1038/s41598-024-75140-7},
year = {2024},
date = {2024-11-06},
abstract = {Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations. |
A bacterial immunity protein directly senses two disparate phage proteins Zhang, Tong In: 2024. @article{noKey,
title = {A bacterial immunity protein directly senses two disparate phage proteins},
author = {Zhang, Tong},
url = {https://www.nature.com/articles/s41586-024-08039-y},
doi = {https://doi.org/10.1038/s41586-024-08039-y},
year = {2024},
date = {2024-10-16},
abstract = {Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers. |
A single Leishmania adenylate forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA J. Jezewski, Andrew In: 2024. @article{noKey,
title = {A single Leishmania adenylate forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA},
author = {J. Jezewski, Andrew},
url = {https://www.jbc.org/article/S0021-9258(24)02381-0/fulltext},
doi = {https://doi.org/10.1016/j.jbc.2024.107879},
year = {2024},
date = {2024-10-09},
abstract = {Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease. |
Targeting RSV-neutralizing B cell receptors with anti-idiotypic antibodies C. Scharffenberger, Samuel In: 2024. @article{noKey,
title = {Targeting RSV-neutralizing B cell receptors with anti-idiotypic antibodies},
author = {C. Scharffenberger, Samuel},
url = {https://www.cell.com/cell-reports/fulltext/S2211-1247(24)01162-8},
doi = {https://doi.org/10.1016/j.celrep.2024.114811},
year = {2024},
date = {2024-10-08},
abstract = {Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies. |
Structure-Based Engineering of Monoclonal Antibodies for Improved Binding to Counteract the Effects of Fentanyl and Carfentanil Rodarte, Justas In: 2024. @article{noKey,
title = {Structure-Based Engineering of Monoclonal Antibodies for Improved Binding to Counteract the Effects of Fentanyl and Carfentanil},
author = {Rodarte, Justas},
url = {https://pubs.acs.org/doi/full/10.1021/acsomega.4c06617},
doi = {https://doi.org/10.1021/acsomega.4c06617},
year = {2024},
date = {2024-10-07},
abstract = {The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest. |
Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus I. Sotiropoulou, Anastasia In: 2024. @article{noKey,
title = {Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus},
author = {I. Sotiropoulou, Anastasia},
url = {https://journals.iucr.org/d/issues/2024/10/00/gm5108/index.html},
doi = {https://doi.org/10.1107/S2059798324009252},
year = {2024},
date = {2024-10-01},
abstract = {β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles. |
Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions Ishii, Kenta In: 2024. @article{noKey,
title = {Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions},
author = {Ishii, Kenta},
url = {https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.17277},
doi = {https://doi.org/10.1111/febs.17277},
year = {2024},
date = {2024-09-26},
abstract = {Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. |
Data driven techniques for the analysis of oral dosage drug formulations Cao, Ziyi In: 2024. @article{noKey,
title = {Data driven techniques for the analysis of oral dosage drug formulations},
author = {Cao, Ziyi},
url = {https://hammer.purdue.edu/articles/thesis/DATA_DRIVEN_TECHNIQUES_FOR_THE_ANALYSIS_OF_ORAL_DOSAGE_DRUG_FORMULATIONS/24142605?file=42411960},
doi = {https://doi.org/10.25394/PGS.24142605.v1},
year = {2024},
date = {2024-09-20},
abstract = {This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning. Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade. Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development. Data acquisition is expensive in the realm of pharmaceutical development, and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such data driven measurements. Among multiple measurement techniques, chemical imaging is an informative tool for analyzing oral drug formulations. However, chemical imaging can often fall into data deprived situations, where data could be limited from the time-consuming sample preparation or related chemical synthesis. An integrated imaging approach, which folds data science techniques into chemical measurements, could lead to a future of informative and cost-effective data driven measurements. In this thesis, the development of data driven chemical imaging techniques for the analysis of oral drug formulations via Fourier transformation and generative adversarial learning are elaborated. Chapter 1 begins with a brief introduction of current techniques commonly implemented within the pharmaceutical industry, their limitations, and how the limitations are being addressed. Chapter 2 discusses how Fourier transform fluorescence recovery after photobleaching (FT-FRAP) technique can be used for monitoring the phase separated drug-polymer aggregation. Chapter 3 follows the innovation presented in Chapter 1 and illustrates how analysis can be improved by incorporating diffractive optical elements in the patterned illumination. While previous chapters discuss dynamic analysis aspects of drug product formulation, Chapter 4 elaborates on the innovation in composition analysis of oral drug products via use of novel generative adversarial learning methods for linear analyses.},
keywords = {FRAP},
pubstate = {published},
tppubtype = {article}
}
This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning. Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade. Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development. Data acquisition is expensive in the realm of pharmaceutical development, and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such data driven measurements. Among multiple measurement techniques, chemical imaging is an informative tool for analyzing oral drug formulations. However, chemical imaging can often fall into data deprived situations, where data could be limited from the time-consuming sample preparation or related chemical synthesis. An integrated imaging approach, which folds data science techniques into chemical measurements, could lead to a future of informative and cost-effective data driven measurements. In this thesis, the development of data driven chemical imaging techniques for the analysis of oral drug formulations via Fourier transformation and generative adversarial learning are elaborated. Chapter 1 begins with a brief introduction of current techniques commonly implemented within the pharmaceutical industry, their limitations, and how the limitations are being addressed. Chapter 2 discusses how Fourier transform fluorescence recovery after photobleaching (FT-FRAP) technique can be used for monitoring the phase separated drug-polymer aggregation. Chapter 3 follows the innovation presented in Chapter 1 and illustrates how analysis can be improved by incorporating diffractive optical elements in the patterned illumination. While previous chapters discuss dynamic analysis aspects of drug product formulation, Chapter 4 elaborates on the innovation in composition analysis of oral drug products via use of novel generative adversarial learning methods for linear analyses. |
Elucidating signal transduction in multi-domain BLUF photoreceptors by studying the Photoactivated Adenylate Cyclase OaPAC Chretien, Anaïs In: 2024. @article{noKey,
title = {Elucidating signal transduction in multi-domain BLUF photoreceptors by studying the Photoactivated Adenylate Cyclase OaPAC},
author = {Chretien, Anaïs},
url = {https://ediss.sub.uni-hamburg.de/handle/ediss/11163},
doi = {null},
year = {2024},
date = {2024-09-13},
abstract = {Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools |
The Human T-cell Leukemia Virus capsid protein is a potential drug target Yu, Ruijie In: 2024. @article{noKey,
title = {The Human T-cell Leukemia Virus capsid protein is a potential drug target},
author = {Yu, Ruijie},
url = {https://www.biorxiv.org/content/10.1101/2024.09.09.612167v1.abstract},
doi = {https://doi.org/10.1101/2024.09.09.612167},
year = {2024},
date = {2024-09-10},
abstract = {Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. |
The toxicity of alpha-syn condensates in the presence and absence of metal ions Pinto, Miriam In: 2024. @article{noKey,
title = {The toxicity of alpha-syn condensates in the presence and absence of metal ions},
author = {Pinto, Miriam},
url = {https://repositorio.ulisboa.pt/handle/10400.5/95870},
doi = {http://hdl.handle.net/10400.5/95870},
year = {2024},
date = {2024-09-08},
abstract = {Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD. |
Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env Agrawal, Parul In: 2024. @article{noKey,
title = {Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env},
author = {Agrawal, Parul},
url = {https://journals.asm.org/doi/epub/10.1128/jvi.00744-24},
doi = {https://doi.org/10.1128/jvi.00744-24},
year = {2024},
date = {2024-09-06},
abstract = {VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.},
keywords = {NT8},
pubstate = {published},
tppubtype = {article}
}
VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. |
