Biology subjects

Williams, K. L.

Publications and source records attributed to Williams, K. L..

9 recordsLinked to original sources

bioRxiv2025

Structural characterization of MAM01 and other cost-effective engineered monoclonal antibodies for malaria prevention

Long-lasting and effective vaccines or monoclonal antibodies (mAbs) for malaria prevention are highly beneficial for people of all ages living in malaria-endemic regions. Recently, two highly protective human mAbs AB000224 (IGHV3-49/IGLV1-40) and AB007088 (IGHV3-33/IGKV1-5), which are encoded by different germline genes, were re-engineered and renamed as MAM01 and MS-1805, respectively, to improve their half-life, developability (including manufacturing), and cost-effectiveness according to WHO guidelines. MAM01 has completed phase 1 and 1b clinical trials (safety / PK / challenge) in US healthy naive adults and is in phase 1 trials (age-de-escalation studies) in Uganda. Here, we determined crystal structures of the antigen-binding fragments (Fabs) of engineered MAM01, MS-1805, and 7088 in complex with different regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP), including junctional, minor, and major repeat regions. Notably, Fab 7088 features an extended CDRL3 comprising 10 amino acids (CDRL3:10) instead of the typical 8 amino acid CDRL3 (CDRL3:8) for VH3-33/VK1-5-encoded mAbs, revealing unique folding within its germline context. Structural comparisons showed that engineered antibodies retain the key molecular and hydrogen bond interactions with no significant conformational changes or loss in binding affinity. These findings demonstrate that the efficacy and affordability of human mAbs can be enhanced by selectively mutating residues in antibody framework regions without compromising binding affinity or epitope interaction.

biophysics↗

bioRxiv2022

Molecular dynamics analysis of Superoxide Dismutase 1 mutations suggests decoupling between mechanisms underlying ALS onset and progression

Mutations in the superoxide dismutase 1 (SOD1) gene are the second most common known cause of ALS. SOD1 variants express high phenotypic variability and over 200 have been reported in people with ALS. Investigating how different SOD1 variants affect the protein dynamics might help in understanding their pathogenic mechanism and explaining their heterogeneous clinical presentation. It was previously proposed that variants can be broadly classified in two groups, wild-type like (WTL) and metal binding region (MBR) variants, based on their structural location and biophysical properties. MBR variants are associated with a loss of SOD1 enzymatic activity. In this study we used molecular dynamics and large clinical datasets to characterise the differences in the structural and dynamic behaviour of WTL and MBR variants with respect to the wild-type SOD1, and how such differences influence the ALS clinical phenotype. Our study identified marked structural differences, some of which are observed in both variant groups, while others are group specific. Moreover, applying graph theory to a network representation of the proteins, we identified differences in the intramolecular contacts of the two classes of variants. Finally, collecting clinical data of approximately 500 SOD1 ALS patients carrying variants from both classes, we showed that the survival time of patients carrying an MBR variant is generally longer (~6 years median difference, p < 0.001) with respect to patients with a WTL variant. In conclusion, our study highlights key differences in the dynamic behaviour of the WTL and MBR SOD1 variants, and wild-type SOD1 at an atomic and molecular level. We identified interesting structural features that could be further investigated to explain the associated phenotypic variability. Our results support the hypothesis of a decoupling between mechanisms of onset and progression of SOD1 ALS, and an involvement of loss-of-function of SOD1 with the disease progression.

neuroscience↗

bioRxiv2022

Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9orf72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 41 genotypically diverse ALS cases with or without FTD, including five cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, and two cases with p.T487I). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=20), unknown familial (n=3), SOD1-linked (n=1), FUS-linked (n=1), C9orf72-linked (n=5), and UBQLN2-linked (n=5) cases. We differentiate between i) ubiquilin 2 aggregation together with pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 gene mutations that cause ALS/FTD. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

neuroscience↗

bioRxiv2021

Super-immunity by broadly protective nanobodies to sarbecoviruses

Vaccine boosters and infection can facilitate the development of SARS-CoV-2 antibodies with improved potency and breadth. Here, we observed super-immunity in a camelid extensively immunized with the SARS-CoV-2 receptor-binding domain (RBD). We rapidly isolated a large repertoire of specific ultrahigh-affinity nanobodies that bind strongly to all known sarbecovirus clades using integrative proteomics. These pan-sarbecovirus nanobodies (psNbs) are highly effective against SARS-CoV and SARS-CoV-2 variants including the Omicron, with the best median neutralization potency at single-digit ng/ml. Structural determinations of 13 psNbs with the SARS-CoV-2 spike or RBD revealed five epitope classes, providing insights into the mechanisms and evolution of their broad activities. The highly evolved psNbs target small, flat, and flexible epitopes that contain over 75% of conserved RBD surface residues. Their potencies are strongly and negatively correlated with the distance of the epitopes to the receptor binding sites. A highly potent, inhalable and bispecific psNb (PiN-31) was developed. Our findings inform on the development of broadly protective vaccines and therapeutics. One sentence summarySuccessive immunization of SARS-CoV-2 RBD in a camelid enhanced the development of super-immunity and isolation and systematic characterization of a large repertoire of ultrahigh-affinity pan-sarbecovirus single-chain VHH antibodies to understand the evolution of this potent and broad immune response.

microbiology↗

bioRxiv2020

SFPQ intron retention, reduced expression and aggregate formation in central nervous system tissue are pathological features of amyotrophic lateral sclerosis

BackgroundSplicing factor proline and glutamine rich (SFPQ, also known as polypyrimidine tract-binding protein-associated-splicing factor, PSF) is a RNA-DNA binding protein with roles in key cellular pathways such as DNA transcription and repair, RNA processing and paraspeckle formation. Dysregulation of SFPQ is emerging as a common pathological feature of multiple neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Increased retention of SFPQ intron nine and nuclear loss of the protein have been linked to multiple genetic subtypes of ALS. Consequently, SFPQ dysregulation has been hypothesised to be a common pathological feature of this highly heterogeneous disease. MethodsThis study provides a comprehensive assessment of SFPQ pathology in large ALS patient cohorts. SFPQ gene expression and intron nine retention were examined in multiple neuroanatomical regions and blood from ALS patients and control individuals using RNA sequencing (RNA-Seq) and quantitative PCR (RT-qPCR). SFPQ protein levels were assessed by immunoblotting of patient and control motor cortex and SFPQ expression pattern was examined by immunofluorescent staining of patient and control spinal cord sections. Finally, whole-genome sequencing data from a large cohort of sporadic ALS patients was analysed for genetic variation in SFPQ. ResultsSFPQ intron nine retention was significantly increased in ALS patient motor cortex. Total SFPQ mRNA expression was significantly downregulated in ALS patient motor cortex but not ALS patient blood, indicating tissue specific SFPQ dysregulation. At the protein level, nuclear expression of SFPQ in both control and patient spinal motor neurons was highly variable and nuclear depletion of SFPQ was not a consistent feature in our ALS cohort. However, we did observe SFPQ-positive cytoplasmic ubiquitinated protein aggregates in ALS spinal motor neurons. In addition, our genetic screen of ALS patients identified two novel, and two rare sequence variants in SFPQ not previously reported in ALS. ConclusionsThis study shows that dysregulation of SFPQ is a feature of ALS patient central nervous system tissue. These findings confirm SFPQ pathology as a feature of ALS and indicate that investigations into the functional consequences of this pathology will provide insight into the biology of ALS.

neuroscience↗

bioRxiv2020

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum

The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I {beta}- and Asn pseudo 310 turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.

microbiology↗

bioRxiv2019

TRIBES: A user-friendly pipeline for relatedness detection and disease gene discovery

SummaryTRIBES is a user-friendly pipeline for relatedness detection in genomic data. TRIBES is the first tool which is both accurate up to 7th degree relatives (e.g. third cousins) and combines essential data processing steps into a single user-friendly pipeline. Furthermore, using a proof-of-principle cohort comprising amyotrophic lateral sclerosis cases with known relationship structures and a known causal mutation in SOD1, we demonstrated that TRIBES can successfully uncover disease susceptibility loci. TRIBES has multiple applications in addition to disease gene mapping, including sample quality control in genome wide association studies and avoiding consanguineous unions in family planning.\n\nAvailabilityTRIBES is freely available on GitHub: https://github.com/aehrc/TRIBES/\n\nContactnatalie.twine@csiro.au\n\nSupplementary informationXXXX

bioinformatics↗

bioRxiv2019

IBD analysis of Australian amyotrophic lateral sclerosis SOD1-mutation carriers identifies five founder events and links sporadic cases to existing ALS families

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by the loss of upper and lower motor neurons resulting in paralysis and eventual death. Approximately 10% of ALS cases have a family history of disease, while the remaining cases present as apparently sporadic. Heritability studies suggest a significant genetic component to sporadic ALS, and although most sporadic cases have an unknown genetic etiology, some familial ALS mutations have also been found in sporadic cases. This suggests that some sporadic cases may be unrecognised familial cases with reduced disease penetrance. Identifying a familial basis of disease in apparently sporadic ALS cases has significant genetic counselling implications for immediate relatives. A powerful strategy to uncover a familial link is identity-by-descent (IBD) analysis which detects genomic regions that have been inherited from a common ancestor. We performed IBD analysis on 90 Australian familial ALS cases from 25 families and three sporadic ALS cases, each of whom carried one of three SOD1 mutations (p.I114T, p.V149G and p.E101G). We identified five unique haplotypes that carry these mutations in our cohort, indicative of five founder events. This included two different haplotypes that carry SOD1 p.I114T, where one haplotype was present in one sporadic case and 20 families, while the second haplotype was found in the remaining two sporadic cases and one family, thus linking these familial and sporadic cases. Furthermore, we linked two families that carry SOD1 p.V149G and found that SOD1 p.E101G arose independently in each family that carries this mutation.

genetics↗

bioRxiv2018

Identification of Kinase Inhibitors that Regulate Nuclear Receptor Nurr1 (NR4A2) Cellular Activity

The ability to regulate the activity NR4A subfamily of nuclear receptors would be potentially useful in the treatment of multiple diseases, but to date, few regulators have been reported. This is likely due to the fact that the NR4A subfamily does not have a typical unoccupied nuclear receptor ligand binding pocket, but rather a pocket that is occupied with hydrophobic side chains of adjacent amino acids. It follows that traditional nuclear receptor assays that seek to identify ligands that bind within the ligand binding pocket would not be successful. We have thus focused on an alternate assay to identify NR4A regulators based on the fact that regulation of NR4A, at least partially, results from phosphorylation/dephosphorylation of the amino terminal region of the protein. We developed a medium throughput cellular assay using a fusion of the amino terminus of Nurr1 (NR4A2) with luciferase reporter and used the assay to screen a large and diverse protein kinase inhibitor set (PKIS). We identified multiple kinase inhibitor compounds from PKIS that significantly increased or decreased the cellular activity of Nurr1. These molecules serve as starting points to discover selective tools for regulation of Nurr1/Nurr1pathway.

pharmacology and toxicology↗