Biology subjects

Lamb, M.

Publications and source records attributed to Lamb, M..

10 recordsLinked to original sources

bioRxiv2025

Population genetic assessment of marine megafauna using seawater environmental DNA: a case study of whale sharks from the Ningaloo Coast World Heritage Area.

There is a growing need for scalable and cost-effective genetic monitoring tools to support the management of species and populations of conservation importance, driven by the increasing biodiversity loss, need for long-term population data and limitations of invasive genetic sampling. Whale sharks (Rhincodon typus), along with other Endangered, elusive and highly migratory species, spend the majority of their lives in offshore ocean waters. This behaviour poses logistical and ethical challenges to invasive genetic sampling of the species, which is traditionally done via tissue biopsy. Here, we develop a genetic toolkit to study populations of whale sharks from seawater environmental DNA using short segments of nuclear DNA (100-300bp) containing two or more SNPs called "microhaplotypes". Amplifying these markers from seawater collected in 1L bottles behind sharks showed that we can reliably genotype sharks from water samples taken immediately behind the animal. Moreover, we observed a strong relationship between population-level allele frequencies and estimates of genetic diversity between eDNA and tissue-derived samples, demonstrating the capability of eDNA in capturing population-level genetic information with high fidelity. We also analysed a tissue dataset of 72 sharks that attended the Ningaloo Coast World Heritage Area over six-years to showcase the utility of our SNP panel to study temporal processes in these populations. Our data revealed patterns of genetic variation through time that were consistent with whole genome techniques. Contributor estimation from mock environmental samples using various combinations of tissue-derived sequence data to estimate abundance of animals in a mixed DNA sample was shown to accurately identify the number of contributors in mixtures containing [≤] 10 individuals, beyond which biases were too large. Our findings illustrate the viability of using microhaplotype markers from seawater eDNA as a tool to study conservation genetics of whale sharks, with the potential for broader expansion of eDNA-based genetic assessments to other marine megafauna and aquatic species.

genetics↗

bioRxiv2025

Solubilization of Membrane Proteins using designed protein WRAPS

The development of therapies and vaccines targeting integral membrane proteins has been complicated by their extensive hydrophobic surfaces, which can make production and structural characterization difficult. Here we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, and function using genetically encoded de novo protein WRAPs (Water-soluble RFdiffused Amphipathic Proteins) that surround the lipid-interacting hydrophobic surfaces, rendering them stable and water-soluble without the need for detergents. We design WRAPs for both beta-barrel outer membrane and helical multi-pass transmembrane proteins, and show that the solubilized proteins retain the binding and enzymatic functions of the native targets with enhanced stability. Syphilis vaccine development has been hindered by difficulties in characterizing and producing the outer membrane protein antigens; we generated soluble versions of four Treponema pallidum outer membrane beta barrels which are potential syphilis vaccine antigens. A 4.0 [A] cryo-EM map of WRAPed TP0698 is closely consistent with the design model. WRAPs should be broadly useful for facilitating biochemical and structural characterization of integral membrane proteins, enabling therapeutic discovery by screening against purified soluble targets, and generating antigenically intact immunogens for vaccine development.

biochemistry↗

bioRxiv2025

NR6A1 is essential for neural crest cell specification, formation and survival

Neural crest cells (NCC) are a migratory progenitor cell population considered unique to vertebrates. Derived from the neuroepithelium during early embryogenesis, NCC contribute to nearly every tissue and organ system throughout the body, and disruptions in NCC development can result in congenital disorders, termed neurocristopathies. Despite decades of research, we have a poor understanding of the cellular mechanisms and signals that govern mammalian NCC formation. We discovered nuclear receptor superfamily 6 group member 1 (NR6A1/GCNF/RTR), is a novel, critical regulator of mammalian NCC specification, formation and survival. Nr6a1 is expressed throughout the neuroepithelium in mouse embryos from E8.0 to E9.5 and briefly in newly delaminated NCC. Nr6a1 loss-of-function perturbs anterior cranial NCC formation and survival, and results in the complete agenesis of migratory NCC caudal to the first pharyngeal arch. Using mouse ESC and human iPSC differentiation into NCC, chromatin immunoprecipitation, and multiomic approaches, we demonstrate that these phenotypes are associated with perturbation of NCC specification (Foxd3, Sox9, Sox10) and epithelial-mesenchymal transition (EMT; Snail1, Zeb2), in concert with persistent expression of pluripotency-associated factors (Oct4 and Nanog) in the neuroepithelium. Conditional deletion revealed that Nr6a1 is required during mid-late gastrulation, demonstrating that murine NCC specification occurs earlier than previously thought. Consistent with these observations, in vivo overexpression of Oct4 in gastrulating mouse embryos disrupts NCC specification and formation. Therefore, Nr6a1 is essential for mammalian NCC formation and moreover, may function as a bimodal switch repressing pluripotency-associated factors in the neuroepithelium, while concomitantly activating NCC specifiers and regulators of EMT.

developmental biology↗

bioRxiv2024

Accurate de novo design of high-affinity protein binding macrocycles using deep learning

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate KD between 1-10 M, and the best anti-GABARAP macrocycle binds with a KD of 6 nM and a sub-nanomolar IC50 in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with KD < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 [A] to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

biochemistry↗

bioRxiv2023

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

biochemistry↗

bioRxiv2023

Computational design of non-porous, pH-responsive antibody nanoparticles

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

bioengineering↗

bioRxiv2023

Design of stimulus-responsive two-state hinge proteins

Proteins that switch between two structural states as a function of environmental stimuli are widespread in nature. These proteins structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Engineering challenges ranging from biological computing devices to molecular motors require such two-state switches, but designing these is an unsolved problem as it requires sculpting an energy landscape with two low-energy but structurally distinct conformations that can be modulated by external inputs. Here we describe a general design approach for creating "hinge" proteins that populate one distinct state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, and double electron-electron resonance spectroscopy demonstrate that despite the significant structural differences, the two states are designed with atomic level accuracy. The kinetics and thermodynamics of effector binding can be finely tuned by modulating the free energy differences between the two states; when this difference becomes sufficiently small, we obtain bistable proteins that populate both states in the absence of effector, but collapse to a single state upon effector addition. Like the transistor, these switches now enable the design of a wide array of molecular information processing systems.

biophysics↗

bioRxiv2023

Design of amyloidogenic peptide traps

Segments of proteins with {beta}-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. Here we describe a general approach to bind such segments in {beta}-strand and {beta}-hairpin conformations using de novo designed scaffolds that contain deep peptide binding clefts flanked by {beta}-strands that form hydrogen bonds to the peptide upon binding. The designs bind their cognate peptides in vitro with nanomolar affinities and in mammalian cells. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide binding cleft is protected in the apo state. We use the approach to design binders to segments of the amyloid forming proteins Transthyretin, Tau, Serum amyloid A1 and A{beta}42. The A{beta} binders block assembly of A{beta} fibrils as effectively as the most potent of the clinically tested antibodies to date.

synthetic biology↗

bioRxiv2022

Precisely patterned nanofibers made from extendable protein multiplexes

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha helical coiled coils have such symmetric extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here, we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibers can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibers can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.

bioengineering↗

bioRxiv2022

Lung Tumor Microphysiological System with 3D Endothelium to Evaluate Modulators of T-Cell Infiltration

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor on chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells infiltrate; and a chemoattractant/tumor compartment. Here, activated T-cells extravasate and infiltrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips, increases T-cell expression of CXCR3 and CXCR4 receptors, and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose a translational strategy by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

bioengineering↗