Biology subjects

Schreiber, A.

Publications and source records attributed to Schreiber, A..

12 recordsLinked to original sources

bioRxiv2026

Sample preparation for direct nanopore based protein identification via tryptic peptide recognition

Rapid, cost-efficient and miniaturized protein analysis is essential for point-of-care clinical diagnostics. Nanopores offer a promising alternative to mass spectrometry, offering high-resolution, single-molecule sensing with a minimal footprint. However, peptide-level protein identification remains challenging due to difficulties in assigning nanopore signals to peptide sequences and the incompatibility of conventional proteolysis workflows with nanopore sensing. Here we integrate a single-pot, solid-phase-enhanced sample preparation (SP3) workflow with nanopore sensing. The SP3 protocol decouples proteolysis from interfering components via magnetic bead cleanup, yielding a digest compatible with nanopore sensing. As a proof of concept, tryptic digests of myelin basic protein measured with an engineered aerolysin variant (AeL-R220S) generated a reproducible five-peak peptide fingerprint. Cross-validation against a synthetic peptide library and LC-MS/MS enabled reference-based assignment of blockade signatures to four MBP-derived peptides. This approach establishes SP3 as a sample-preparation workflow for nanopore sensing, paving the way for label-free identification and characterization of protein targets.

biochemistry↗

bioRxiv2026

Phosphoregulated SMCR8-FIP200 interaction connects the ALS/FTD-linked C9orf72 complex to autophagy initiation and mitochondrial quality control

Hexanucleotide (GGGGCC) repeat expansions in the non-coding region of C9ORF72 are a major genetic cause of ALS/FTD and reduce C9orf72-SMCR8-WDR41 complex levels, but how this contributes to autophagy-lysosome dysfunction and previously reported mitochondrial quality-control defects in C9ORF72-ALS/FTD remains unclear. Here we identify a direct interaction between SMCR8 and the FIP200 subunit of the ULK1/2 autophagy initiation complex, mediated by two FIP200-interacting region (FIR) motifs in a disordered SMCR8 loop. Phosphorylation of these motifs by ULK1/2 or TBK1 strengthens binding and promotes ULK1/2 complex association in cells. Stabilising the SMCR8-FIP200 interaction suppresses Parkin-dependent mitophagy, whereas both stabilisation and weakening impair deferiprone-induced mitophagy, while leaving bulk autophagy, lysophagy and ivermectin-induced mitophagy largely intact. These findings define a regulated C9orf72-ULK1/2 axis and provide a mechanistic framework by which repeat-expansion-associated reduction in C9orf72 complex abundance may contribute to previously observed mitochondrial quality-control defects in C9ORF72-ALS/FTD.

cell biology↗

bioRxiv2025

A structural and regulatory framework for Atg9-containing vesicle formation and their Atg1-dependent remodelling during autophagy initiation

Autophagy is a complex intracellular degradation pathway that depends on the coordinated interplay between the core autophagy machinery and diverse membrane sources to drive the de novo formation of double-membrane vesicles, known as autophagosomes. Golgi-derived Atg9-containing vesicles are essential for this process, delivering membranes to the pre-autophagosomal structure (PAS). These vesicles contain the transmembrane proteins Atg9 and Atg27 and the peripheral membrane protein Atg23; however, the nature, function, and regulation of their interactions remain poorly understood. Here, we systematically dissect the molecular interactions between Atg9, Atg23 and Atg27, and uncover their regulation in space and time. The bipartite binding mode by which Atg23 engages Atg9 provides a structural model for how Atg23 promotes vesicle budding. Furthermore, Atg1-dependent phosphorylation of Atg9 remodels its interactions with Atg23 and Atg27 at the PAS to support autophagy initiation. Together, these findings establish a molecular and regulatory framework for the earliest steps of autophagy.

biochemistry↗

bioRxiv2024

Mechanistic studies of autophagic cargo recruitment and membrane expansion through in vitro reconstitution

Autophagy is a highly conserved catabolic pathway to remove deleterious cytosolic material to maintain cellular homeostasis and cell survival. Upon autophagy induction, a unique double-membraned structure, called a phagophore, forms and expands into a cup shape to engulf these cytosolic substrates. ATG8 proteins are covalently conjugated to autophagic membranes by lipidation of phosphatidylethanolamine (PE) and are thought to localise on both sides of the phagophore membrane. ATG8 conjugated on the inner membrane of the phagophore recruits autophagy cargo receptors, such as p62. To recapitulate events on the inner membrane, we used giant unilamellar vesicles (GUVs) as a model membrane and encapsulated proteins of interest inside GUVs, thus generating a membrane platform to which ATG8 proteins could be localised on the inner leaflet of the vesicles. We reconstituted WIPI2b-directed and cargo-directed ATG8 lipidation inside the GUVs and revealed distinct roles of WIPI2b and p62 in initiating the ATG conjugation cascade. Furthermore, we showed that p62 or p62 droplets were recruited to the inner membrane of the GUVs though interaction with membrane-bound ATG8s. Using a bead-based membrane expansion assay, we demonstrated a redistribution and local enrichment of membrane-bound ATG8s across the membrane upon interaction with p62 and p62 droplets. Our study provides novel model systems to investigate the interactions on the inner membrane of the phagophore and reveals fundamental molecular insights into phagophore membrane bending. This process is directed by ATG8-cargo interaction, during which cargo receptors concentrate ATG8 proteins on the inner surface of the phagophore membrane.

biochemistry↗

bioRxiv2024

The challenge of sequencing Chlamydia trachomatis and other bacterial STI genomes directly from clinical swabs: the optimum solution

Rates of bacterial sexually transmitted infections (STIs) are rising and accessing their genomes provides information on strain evolution, circulating strains, and encoded antimicrobial resistance (AMR). Notable pathogens include Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP), globally the most common bacterial STIs. Mycoplasma genitalium (MG) is also a bacterial STI which is of concern due to AMR development. These bacteria are also fastidious or hard to culture, and standard sampling methods lyse bacteria, completely preventing pathogen culture. Clinical samples contain large amounts of human and other microbiota DNA. These factors hinder the sequencing of bacterial STI genomes. We aimed to overcome these challenges in obtaining whole genome sequences, and evaluated four approaches using clinical samples from Argentina (39), Switzerland (14), and cultured samples from Finland (2) and Argentina (1). First, direct genome sequencing from swab samples was attempted through Illumina deep metagenomic sequencing, showing extremely low levels of target DNA, with under 0.01% of the sequenced reads being from the target pathogens. Second, host DNA depletion followed by Illumina sequencing was not found to produce enrichment in these very low load samples. Third, we tried a selective long-read approach with the new adaptive sequencing from Oxford Nanopore Technologies (ONT), which also did not improve enrichment sufficiently to provide genomic information. Finally, target enrichment using a novel pan-genome set of custom SureSelect probes targeting CT, NG, TP, and MG followed by Illumina sequencing was successful. We produced whole genomes from 64% of CT positive samples; from 36% of NG positive samples, and from 60% of TP positive samples. Additionally, we enriched MG DNA to gain partial genomes from 60% of samples. This is the first publication to date to utilize a pan-genome STI panel in target enrichment. Target enrichment, though costly, proved essential for obtaining genomic data from clinical samples. This data can be utilized to examine circulating strains, genotypic resistance, and guide public health strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/624631v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@578aeborg.highwire.dtl.DTLVardef@16185f9org.highwire.dtl.DTLVardef@1a2ae87org.highwire.dtl.DTLVardef@1703fa7_HPS_FORMAT_FIGEXP M_FIG C_FIG Impact statementGenome data on circulating sexually transmitted infections (STIs) is important to better understand transmission networks, antimicrobial resistance and to guide treatment decisions. For many bacterial STIs, this information is difficult to obtain, as the bacteria are fastidious, in some cases intracellular, and often recalcitrant to culture. We have developed and tested a target enrichment STI panel of baits to capture whole genomes of Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma genitalium with approximately 50% success in genome sequencing for the first three pathogens. We compare this against other sequencing and enrichment methods, which did not provide sufficient data for genome analysis. This panel approach shows potential for clinical samples carrying these pathogens and can potentially also be developed for further pathogen groups. Data summaryAll illumina sequence data, with human read data removed using Hostile (1) and KrakenTools (https://github.com/jenniferlu717/KrakenTools), is deposited with the European Nucleotide Archive (ENA) under project number PRJEB72167.

microbiology↗

bioRxiv2024

Lifespan in rodents with MYT1L heterozygous mutation

MYT1L syndrome is a newly recognized disorder characterized by intellectual disability, speech and motor delay, neuroendocrine disruptions, ADHD, and autism. In order to study this gene and its association with these phenotypes, our lab recently created a Myt1l heterozygous mutant mouse inspired by a clinically relevant mutation. This model recapitulates several of the physical and neurologic abnormalities seen in humans with MYT1L syndrome, such as weight gain, microcephaly, and behavioral disruptions. The majority of patients with this syndrome are young, and little is known about the impact of age on health and mortality in these patients. Using a Myt1l mutant mouse, we examined the impact of Myt1l mutation on body weights, lifespan, and histopathology findings of mice at the end of life. This cohort of heterozygous mice demonstrated increased body weight across the lifespan, however there was no significant difference in lifespan, apparent cause of death, or end of life histopathological findings between Myt1l heterozygous and wildtype mice. These findings suggest while Myt1l heterozygous mutation may influence overall brain development, it does not strongly impact other organ systems in the body over time.

genetics↗

bioRxiv2024

Engineering a novel probiotic toolkit in Escherichia coli Nissle1917 for sensing and mitigating gut inflammatory diseases

Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacterium Escherichia coli Nissle 1917 (EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide, a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNF nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to nitric oxide, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by ELISA and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/599326v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@173d556org.highwire.dtl.DTLVardef@1bb2ae1org.highwire.dtl.DTLVardef@fc1fcborg.highwire.dtl.DTLVardef@8b5f7f_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Table of Contents. The engineered probiotic system: Inflamed intestinal cells release the inflammatory regulator TNF (depicted as red squares), which promotes inflammation through a positive feedback loop. Concurrently, these cells produce large amounts of nitric oxide (NO, represented by yellow circles) during inflammation. Our custom-engineered EcN biosensor can detect NO using a NorR-based sensor (in purple) and subsequently trigger the production of nanobodies (in turquoise). These nanobodies are then released into the extracellular environment via a specially engineered secretion system in the bacterial host (shown in dark blue). Once outside the cell, the nanobodies attach to TNF, effectively sequestering them and reducing inflammation. The graph at the bottom of this panel illustrates the general behavior of our system: nanobody production starts upon reaching a certain NO concentration threshold and continues in an NO-dependent fashion. As nanobodies are produced, they capture TNF, leading to a reduction in inflammation and a decrease in NO production. This decrease in NO then halts the nanobody production. SignificanceProbiotics can be engineered to detect and act upon extracellular disease indicators, optimizing therapeutic outcomes. Particularly, self-regulating sense-and-respond genetic circuits have the potential to enhance the accuracy, efficacy, and adaptability of treatment interventions. In this study, we developed and characterized a new integrated and modular toolkit that detects a gut inflammation biomarker, specifically nitric oxide, and responds to it in an inducible manner by secreting humanized nanobodies targeting the pro-inflammatory molecule TNF. We also develop a coarse-grained mathematical framework for modelling engineered probiotic activity in the gut. This novel system contributes to current efforts to develop new engineered probiotic systems and holds promise for inspiring new treatments for gut inflammation associated with various autoimmune diseases.

synthetic biology↗

bioRxiv2023

ESMBind and QBind: LoRA, QLoRA, and ESM-2 for Predicting Binding Sites and Post Translational Modification

In this study we discuss the viability of applying protein language models to the problem of predicting bindings sites of protein sequences from single sequences alone using Low Rank Adaptation (LoRA) and Quantized Low Rank Adaptation (QLoRA). No Multiple Sequence Alignment (MSA) or structural information for the proteins was used. Moreover, using LoRA and QLoRA shows improved performance over vanilla full finetuning, and significantly helps in mitigating overfitting. Also, due to the efficiency of LoRA and QLoRA, we are able to train the larger ESM-2 models on modest hardware, making the method very attractive and accessible. We also note that this technique serves as an important regularization technique and serves to improve generalization of models on unseen data.

molecular biology↗

bioRxiv2022

Cis-membrane association of human ATG8 proteins N-terminus mediates autophagy

Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. The cis-membrane association of the N-terminus is critical to maintain membrane expansion and the size of autophagosomes in cells, consequently, mediating the efficient degradation of p62. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

cell biology↗

bioRxiv2021

SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids

Several studies have pointed to retinal involvement in COVID-19 disease, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell-derived retinal organoids to study retinal infection by the SARS-CoV-2 virus. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID-19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS-CoV-2 infection of retinal organoids, indicating that SARS-CoV-2 infects retinal cells in an ACE2-dependent manner. These results suggest a retinal involvement in COVID-19 and emphasize the need to monitor retinal pathologies as potential sequelae of "long COVID".

molecular biology↗

bioRxiv2019

High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy

Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel Kv2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant Kv2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type Kv2.1. Quantification of protein expression also identified variants with reduced total Kv2.1 expression or deficient cell-surface expression.\n\nOur study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.

genetics↗

bioRxiv2018

A prebiotic protocell model based on dynamic protein membranes accommodating anabolic reactions

Phospholipid membranes are essential constituents of extant cells rendering them preferred candidates as membrane components in origin of life scenarios. These models greatly neglect stability requirements and their problematic synthetic complexity necessary to access such lipid membrane constituents under early life conditions. Here we present an alternative protocell model, based on amphiphilic protein membranes constituted of prebiotic amino acids. These self-assembled dynamic Protein Membrane Based Compartments (PMBC) are impressively stable and compatible with prevalent protocell membrane constituents. PMBCs can enclose functional proteins, undergo membrane fusion, phase separate, accommodate anabolic ligation reactions and DNA encoded synthesis of their own membrane constituents. Our findings suggest that prebiotic PMBC represent a new type of protocell as plausible ancestor of current lipid-based cells. They can be used to design simple artificial cells important for the study of structural and catalytic pathways related to the emergence of life.

evolutionary biology↗