Biology subjects

Buckley, S.

Publications and source records attributed to Buckley, S..

8 recordsLinked to original sources

bioRxiv2026

A generalizable interface-seeded framework for de novo design of functional oligomers

Protein oligomers are ubiquitous in biological systems and essential for function. However, the de novo design of oligomers that controllably assemble in response to exogenous stimuli remains challenging. Here, we present an AI-based generative approach that leverages an interface-seeded strategy for designing responsive homo-oligomers from isolated interaction modules. Experimentally validated designs are highly accurate and explore new-to-nature topologies. We show that designs effectively respond to their chemical triggers with conditional oligomerization or to phosphorylation-driven conformational changes with reversible oligomerization. We further functionalized our responsive assemblies to build ligand-dependent membrane binding systems and phosphorylation-controlled gene regulatory switches. Our framework enables the generalizable design of responsive protein complexes, opening novel possibilities for the engineering of biosynthetic systems with sophisticated regulatory mechanisms.

biochemistry↗

bioRxiv2025

Rational design of chemically responsive cytokines for cancer immunotherapy

Cytokines are key signal mediators of the immune system, playing essential roles in regulating central immunological processes. Despite their unique ability to modulate the immune system, the translation of cytokine-based therapies to the clinic has been significantly hindered by severe toxicities resulting from the pleiotropy and off-target effects of many cytokines. Here, we present a general strategy for the design of switchable cytokines that enables precise control over cytokine activity using the clinically approved drug, Venetoclax. We rationally designed self-inhibited Switchable InterLeukins (SwILs) by embedding the chemically controlled interface (Bcl-2: BIM-BH3) into the cytokines structure, resulting in conditional cytokines activated by Venetoclax. We showed the broad applicability of this strategy across various cytokines, including IL-2, IL-15, and IL-10. In vivo studies with IL-15 demonstrated that the SwILs achieved Venetoclax-dependent tumor control comparable to that of the native cytokine. Additionally, we showed that this strategy can be expanded to respond to other tumor-intrinsic stimuli, such as tumor-specific proteases. Overall, SwILs offer control of cytokine activity, enhancing the safety and clinical applicability of cytokine-based therapeutics.

bioengineering↗

bioRxiv2025

De novo design of phosphorylation-induced protein switches for synthetic signaling in cells

A hallmark of living systems is their ability to respond and adapt to many types of exogenous and endogenous cues. At the core of this capability lie the evolved mechanisms of cellular signaling which perform the relay of biochemical signals throughout the cell. Much of this core signaling functionality in living systems is afforded by the ability of proteins to change their chemical and structural "status", by populating distinct states in conformational landscapes, undergoing post-translational modifications that alter these populations, and dynamically interacting with other cellular partners. Despite the remarkable advances we have witnessed in computational protein design, it remains an outstanding challenge to rationally design many of these aspects that are at the core of biological function. Here, we set out to design a minimal signaling network triggered by phosphorylation, which relies on de novo components that sample low-populated conformational states to control a designed protein interaction. In these designed components we implemented principles of signal amplification, conformational heterogeneity and molecular recognition which are ubiquitously used by nature to sustain biological function in cells. The designed proteins were biochemically and structurally characterized and ultimately are functional in cell-based systems where they regulate the transcription of reporter proteins in a phosphorylation-dependent manner. Overall, this work lays the foundation for designing synthetic signaling cascades using de novo protein components, which could be important to enhance our understanding of natural systems and for relevant applications in synthetic biology.

bioengineering↗

bioRxiv2024

BindCraft: one-shot design of functional protein binders

Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold21 to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins, and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity, and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Lastly, we utilize cell surface receptor-specific binders to redirect AAV capsids for targeted gene delivery. This work represents a significant advancement towards a "one design-one binder" approach in computational design, with immense potential in therapeutics, diagnostics, and biotechnology.

bioinformatics↗

bioRxiv2024

Targeting protein-ligand neosurfaces using a generalizable deep learning approach

Molecular recognition events between proteins drive biological processes in living systems. However, higher levels of mechanistic regulation have emerged, where protein-protein interactions are conditioned to small molecules. Here, we present a computational strategy for the design of proteins that target neosurfaces, i.e. surfaces arising from protein-ligand complexes. To do so, we leveraged a deep learning approach based on learned molecular surface representations and experimentally validated binders against three drug-bound protein complexes. Remarkably, surface fingerprints trained only on proteins can be applied to neosurfaces emerging from small molecules, serving as a powerful demonstration of generalizability that is uncommon in deep learning approaches. The designed chemically-induced protein interactions hold the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells.

biochemistry↗

bioRxiv2020

Valorization of CO2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H2 and CO2 and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO2 under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20-60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO2. And, we engineered C. necator to convert CO2 into the LCO, a plant growth enhancer, with titers of [~]1.4 mg/L--equivalent to yields in its native source, Bradyrhizobium. We applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding f how a gas-utilizing bacteria can promote sustainable production.

synthetic biology↗

bioRxiv2019

Pathognomonic and epistatic genetic alterations in B-cell non-Hodgkin lymphoma

B-cell non-Hodgkins lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

genomics↗

bioRxiv2018

Gene therapy in argininosuccinic aciduria

Argininosuccinate lyase (ASL) belongs to the liver-based urea cycle detoxifying ammonia, and the citrulline-nitric oxide cycle synthesising nitric oxide (NO). ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia and a multi-organ disease with neurocognitive impairment. Current therapeutic guidelines aim to control ammonaemia without considering the systemic NO imbalance. Here, we observed a neuronal disease with oxidative/nitrosative stress in ASL-deficient mouse brains. A single systemic injection of gene therapy mediated by an adeno-associated viral vector serotype 8 (AAV8) in adult or neonatal mice demonstrated the long-term correction of the urea cycle and the citrulline-NO cycle in the brain, respectively. The neuronal disease persisted if ammonaemia only was normalised but was dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This was correlated with behavioural improvement and a decrease of the cortical cell death rate. Thus, the cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress not mediated by hyperammonaemia, which is reversed by AAV gene transfer targeting the brain and the liver, acting on two different metabolic pathways via a single vector delivered systemically. This approach provides new hope for hepatocerebral metabolic diseases.

genetics↗