Biology subjects

Lang, K.

Publications and source records attributed to Lang, K..

14 recordsLinked to original sources

bioRxiv2026

Magnetic DNA Origami Nanorotors

Self-assembled DNA nanostructures show great promise as functional devices, highly configurable materials, and in nanorobotics. Magnetic control can provide a powerful actuation mechanism in a broad range of contexts, since it affords a high-level of external control, it is biocompatible, and orthogonal to chemical or electrical stimuli. Here we demonstrate magnetic molecular nanoactuators by leveraging the unique site-specificity of DNA origami to assemble highly anisotropic magnetic nanocubes on high-aspect ratio DNA origami bundles. We traced and controlled 100s of our DNA origami nanorotors at the single-rotor level and demonstrated their programmable magnetic clamping and controlled rotation under uniform and rotating magnetic fields. By varying the population and inter-particle spacing of the nanocubes, magnetic torque values in the order of 10-100 pN nm are achieved at field strengths < 10 mT. Monte Carlo simulations reveal that assembly of nanocubes on DNA origami rotors leads to collective magnetic properties, with numerically estimated torque values in good agreement with the experiments. Our magnetic nanorotors offer a foundation for biocompatible nanorobotics, as well as high-throughput magnetic force and torque tweezers.

biophysics↗

bioRxiv2026

Single-molecule identification of full-length proteins with single-amino-acid resolution using nanopores

Nanopore-based technologies show promise in single-molecule protein sequencing. By using an unfoldase and a nanopore with enhanced electroosmotic flow, here we show the continuous identification of generic proteins during single nanopore passes. This approach enables the recording of differences in charge and size from single amino acid substitutions compared to reference signals, paving the way for single-molecule protein sequencing and high-throughput proteomics.

biophysics↗

bioRxiv2025

The functional landscape of the human ubiquitinome

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. To date, hundreds of thousands of ubiquitination sites (ubi-sites) have been identified, however fewer than 1% have known functional roles. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. To pinpoint critical regulatory events requiring ubiquitination at a precise site, we mapped ubi-site conservation across proteomics data from six non-human species. Perturbation proteomics revealed that highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize site-specific ubiquitination relevant for organismal fitness, we constructed a machine learning-based positional importance score for more than 100,000 ubi-sites, which identifies sites regulating diverse protein functions and rationalizes genetic vulnerabilities. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying site-specific protein ubiquitination.

cell biology↗

bioRxiv2025

A Modular Genetic Code Expansion Approach to Site-Specific Lysine Acylations

Lysine acylations are a diverse class of post-translational modifications (PTMs) that dynamically regulate protein function. Access to homogenous, site-specifically modified proteins is essential for dissecting their molecular roles, yet remains challenging with traditional methods. Here we present a modular strategy that combines genetic code expansion (GCE) with a chemoselective amide bond-forming reaction to install a wide range of lysine acylations directly onto folded proteins. Key to this approach is the genetic encoding of N{varepsilon}-methoxylysine, which reacts efficiently with N-methyliminodiacetyl (MIDA) acylboronates to generate diverse acyl lysine modifications from a single protein precursor, including several acyl PTMs not previously accessible through GCE. This mild and broadly applicable methodology enables systematic studies of lysine acylation on multiple target proteins. We demonstrate its utility by probing the effects of defined acyl modifications on enzymatic activity, protein-RNA interactions and deacylase-mediated regulation. Together, this platform establishes a versatile strategy to interrogate the functional consequences of this wide-spread class of PTMs.

synthetic biology↗

bioRxiv2025

Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W

Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet access to such conjugates remains a major challenge. Existing approaches are often constrained by low yields, harsh reaction conditions, engineered recognition motifs or non-native linkage architectures. Here, we present UbyW (Ubiquitylation by UBE2W), a programmable platform for site-specific ubiquitylation that repurposes the E2 enzyme UBE2W to target genetically encoded isopeptidic neo-N-termini. UbyW enables efficient generation of near-native Ub-protein conjugates across diverse protein substrates, including endogenous ubiquitylation sites within folded domains, and can be implemented through a reconstituted intracellular cascade in Escherichia coli for streamlined high-yield production. The platform further enables installation of chemical functionalities adjacent to the isopeptidic linkage, including photocrosslinkers for capturing modification-dependent interactions. Using programmable probes targeting site-specific ubiquitylation of the small GTPase Ran, we identify USP15 as a cognate deubiquitylase and show that Ran K71 monoubiquitylation disrupts key Ran-cycle interactions.

biochemistry↗

bioRxiv2025

Structural basis for the ubiquitin chain recognition of the human 26S proteasome

Proteasomal degradation is a fundamental process for all eukaryotic life. A protein destined for degradation is first tagged with a polyubiquitin chain, which is selected by the proteasome. Different ubiquitin chain topologies serve as distinct signals, with K48-linked chains acting as the canonical degradation signal and K11/K48-branched chains providing even more potent targeting, particularly during cell cycle regulation. However, the structural basis for how the proteasome distinguishes between these different chain architectures has remained unclear. Here, we present high-resolution cryo-EM structures of the human 26S proteasome bound to both a K48-linked tetraubiquitin chain and a K11/K48-branched chain. Our structures reveal distinct binding modes for these two types of chain linkage. K48 chains wrap around the Ubiquitin interaction motif of the receptor RPN10 in an unexpected spiral conformation, while K11 branches engage the proteasome through previously uncharacterised interfaces in a cleft formed between RPN2 and RPN10. Through structure-guided mutagenesis and cellular studies, we demonstrate that these binding modes are essential for efficient substrate degradation and cell cycle progression. These findings establish how the proteasome achieves selective substrate recognition through chain topology-specific interactions.

biochemistry↗

bioRxiv2025

The slit diaphragm in Drosophila features a bi-layered, fishnet-like architecture

The kidney filters large volumes of blood plasma, relying on the glomerulus for the filtration. The slit diaphragm, a critical component of the glomerulus, is formed between podocytes by the immunoglobulin domain proteins nephrin and Neph1. The molecular architecture of the slit diaphragm has remained elusive for decades. Using cryo-electron tomography on focused ion beam-milled Drosophila nephrocytes, an invertebrate podocyte model, we show that the slit diaphragm adopts a fishnet-like pattern. Comparison of hundreds of slit diaphragm segments reveals that it is bi-layered and highly periodic. Based on the cryo-electron tomography map, we propose four possible models for the arrangement of the Drosophila nephrin ortholog (Sns), and the Drosophila Neph1 ortholog (Kirre), the main components of the slit diaphragm in Drosophila. In each model, precise and consistent homo- and heterophilic interactions between crossing immunoglobulin domains of Sns and Kirre become apparent, with immediate implications for the stability and the assembly of the slit diaphragm. Cryo-electron tomography shows that sns silencing disrupts this fishnet pattern, linking this directly to Drosophila nephrin. After Rab5 silencing, causing Sns mistrafficking and ectopic SD formation, the fishnet pattern appears ectopically as well. Our findings align with observations applying cryo-electron tomography to podocytes in mice, indicating that the molecular architecture is evolutionarily conserved across animals. This highlights the value of the nephrocyte as a podocyte model and establishes a crucial link between the architecture of the slit diaphragm and its function.

biophysics↗

bioRxiv2025

Hijacking a bacterial membrane transporter for efficient genetic code expansion

The site-specific encoding of non-canonical amino acids (ncAAs) provides a powerful tool for expanding the functional repertoire of proteins. Its widespread use for basic research and biotechnological applications is, however, hampered by low efficiencies of current ncAA incorporation strategies. We uncover poor cellular ncAA uptake as a main obstacle to efficient genetic code expansion and overcome this bottleneck by hijacking a bacterial membrane transporter to actively import isopeptide-linked ncAAs within easily synthesizable tripeptide-scaffolds. Using this approach, we enable efficient encoding of eleven previously inaccessible ncAAs, decorating proteins with bioorthogonal and crosslinker moieties, posttranslational modifications, and functionalities for chemoenzymatic conjugation. To enhance scalability of protein production, we evolve the membrane transporter for preferential import of isopeptide-linked tripeptides, creating a novel Escherichia coli strain that facilitates single and multi-site ncAA incorporation with wild type efficiencies. Additionally, we adapt the tripeptide-scaffolds for co-transport of two different ncAAs, enabling their efficient dual incorporation. This work underscores the importance of optimizing ncAA-uptake for high-yielding production of modified proteins and will accelerate the development of generalizable transport systems, aiding incorporation of non-canonical building blocks to broaden the chemical space of proteins without the need to design for passive membrane permeability.

synthetic biology↗

bioRxiv2024

One-Pot Dual Protein Labelling for Simultaneous Mechanical and Fluorescent Readouts in Optical Tweezers

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Forster resonance energy transfer (FRET) pairs, addresses this issue. Moreover, attaching one FRET probe to a tethered protein and the other to a protein in solution allows precise localisation of interaction sites, while probing mechanical properties. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labelling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labelled with the FRET pair maleimide-Atto550 and maleimide-Atto647N, alongside single- stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labelled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing protein conformational dynamics and protein interactions beyond the limitations of traditional optical tweezers. StatementThe developed method combines fluorescence microscopy with optical tweezers, enhancing single-molecule protein studies by overcoming the limitations of one- directional mechanical probing. Utilizing two orthogonal protein conjugation methods for one-pot dual labelling, the heat shock protein 90 was labelled with a FRET pair and single-stranded DNA oligonucleotides. Validated by comparison with published conformational changes, mechanical unfolding signatures, and FRET pair distances, this approach provides a powerful tool to explore single-molecule conformational dynamics and protein interactions.

biophysics↗

bioRxiv2023

CyVerse: Cyberinfrastructure for Open Science

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 110,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in two countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

scientific communication and education↗

bioRxiv2022

Selective endocytosis controls slit diaphragm maintenance and dynamics.

The kidneys generate about 180 liters of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.

cell biology↗

bioRxiv2019

Viral infection overcomes ineffectiveness of anti-tumoral CD8+ T cell mediated cytotoxicity

With the integration of PD-1 and CTLA-4 targeting immune checkpoint blockade into cancer treatment regimes, the anti-tumoral cytotoxicity of tumor-specific CD8+ T cells is well established. However, while the unresponsiveness of CD8+ T cells against big tumors is mainly explained by T cell exhaustion, other factors contributing to CD8+ T cell failure remain not well studied. Here we used a mouse melanoma model to study the interaction of growing tumor cells, innate immunity and CD8+ T cell responses induced by viral replication. Mouse model of melanoma (B16F10-OVA) and infections with arenaviruses. Growing B16F10-OVA cells did not induce systemic ablation of tumor specific CD8+ T cells. However, despite the presence of tumor-infiltrating CD8+ T cells, the anti-tumoral immune response was very limited. T cell anergy against the tumor was accompanied with a strong down-regulation of MHC-I on advanced tumors. LCMV infection restored the MHC class I expression, enhanced T cell function and lead to tumor regression. This study shows that tumor progression does not necessary lead to systemic exhaustion of the anti-tumoral CD8+ T cell response. Lack of innate signals is an additional reason for limited CD8+ T cell mediated cytotoxicity against the tumor.

immunology↗

bioRxiv2018

A Gender-Dependent Molecular Switch of Inflammation via MyD88/Estrogen Receptor-alpha Interaction

Most Toll-like receptors and IL-1/IL-18 receptors activate a signaling cascade via the adaptor molecule MyD88, resulting in NF-{kappa}B activation and inflammatory cytokine and chemokine production. Females are less susceptible than males to inflammatory conditions, presumably due to protection by estrogen. Here we show that MyD88 interacts with a methylated, cytoplasmic form of estrogen receptor-alpha (methER-). This interaction is required for NF-{kappa}B transcriptional activity and pro-inflammatory cytokine production, and is dissociated by estrogen. Importantly, we show a strong gender segregation in gametogenic reproductive organs, with MyD88/methER- interactions found in testicular tissues and in ovarian tissues from menopausal women, but not in ovaries from women age 49 and less -suggesting a role for estrogen in disrupting this complex in situ. Collectively, our results indicate that the formation of MyD88/methER- complexes during inflammatory signaling and their disruption by estrogen may represent a mechanism that contributes to gender bias in inflammatory responses.

immunology↗

bioRxiv2017

Accurate typing of class I human leukocyte antigen by Oxford nanopore sequencing

Oxford Nanopore Technologies MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunological compatibility between organ donors and recipients, but the platforms high error rate makes it challenging to type alleles with clinical-grade accuracy. Here, we developed and validated Athlon, an algorithm that iteratively scores nanopore reads mapped to a hierarchical database of HLA alleles to arrive at a consensus diploid genotype; Athlon achieved a 100% accuracy in class I HLA typing at high resolution.

bioinformatics↗