Biology subjects

Vielmetter, J.

Publications and source records attributed to Vielmetter, J..

5 recordsLinked to original sources

bioRxiv2025

A multiplex extracellular interactome screening method employing high-avidity nanoparticles

Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.

molecular biology↗

bioRxiv2025

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗

bioRxiv2023

Engineering antigen-specific tolerance to an artificial protein hydrogel

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "non-self" artificial protein hydrogels which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the non-self gels raised significantly higher anti-gel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the non-self hydrogel epitopes to DEC-205, an endocytic receptor involved in Treg induction, fully suppressed the elevated antibody titer against the non-self gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

bioengineering↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗

bioRxiv2021

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. Here we report isolation and characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7[A] cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, implying that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

immunology↗