Biology subjects

Murray, J. W.

Publications and source records attributed to Murray, J. W..

15 recordsLinked to original sources

bioRxiv2026

Co-optation of the regenerative role of type 2 alveolar cells in distal lung repair in mice by terminal airway epithelial cells in humans

The lung is endowed with extensive regenerative capacity. The principal cell of the lung is the type 1 alveolar epithelial (AT1) cell, which mediates gas exchange. Mouse models implicated surfactant-producing type 2 alveolar epithelial (AT2) cells as facultative stem cells that regenerate AT1 cells after injury through a transitional KRT8+ intermediate. Larger mammals, however, possess terminal and respiratory bronchioles that are lined by alveoli and by epithelial cells that are absent in mice. Here we show, using human pluripotent stem cell-derived lung organoids and comparative computational analysis, a prime role for terminal and respiratory bronchiole cells in AT1 regeneration without AT2 intermediate. Furthermore, cells similar to aberrant basaloid cells, profibrotic elements that accumulate in pulmonary fibrosis, are physiological intermediates in a more rapidly committing trajectory from terminal and respiratory bronchioles to AT1 cells marked by expression of KRT17, are regulated by Hippo and TGF{beta} signaling, and are transcriptionally distinct from mouse AT2-derived KRT8+ transitional cells. Our findings indicate that terminal and respiratory bronchioles in humans have to a large extent co-opted the regenerative and pathogenic functions of AT2 cells in mice. Efforts to enhance or correct human lung regeneration should therefore focus on facultative airway-derived alveolar progenitors using human models.

cell biology↗

bioRxiv2025

Designing Novel Solenoid Proteins with In Silico Evolution

Solenoid proteins are elongated tandem repeat proteins with diverse biological functions, making them attractive targets for protein design. Advances in machine learning have transformed our understanding of sequence-structure relationships, enabling new approaches for de novo protein design. Here, we present an in silico evolution platform that couples a solenoid discriminator network with AlphaFold2 as an oracle within a genetic algorithm. Starting from random sequences, we design -, {beta}-, and {beta}-solenoid backbones, generating structures that span natural and novel solenoid space. We experimentally characterise 41 solenoid designs, with -solenoids consistently folding as intended, including one structurally validated design that closely matches the design model. All {beta}-solenoids initially failed, reflecting the difficulty of designing {beta}-strand majority proteins. By introducing terminal capping elements and refining designs based on earlier experimental screens, we generate two {beta}-solenoids that have biophysical properties consistent with their designs. Our approach achieves fold-specific hallucination-based design without depending on explicit structural templates.

synthetic biology↗

bioRxiv2025

Exploring the structural diversity and evolution of the D1 subunit of photosystem II using AlphaFold and Foldtree

While our knowledge of photosystem II has expanded to time-resolved atomic details, the diversity of experimental structures of the enzyme remains limited. Recent advances in protein structure prediction with AlphaFold offer a promising approach to fill this gap in structural diversity in non-model systems. This study used AlphaFold to predict the structures of the D1 protein, the core subunit of photosystem II, across a broad range of photosynthetic organisms. The prediction produced high-confidence structures, and structural alignment analyses highlighted conserved regions across the different D1 groups, which were in line with high pLDDT scoring regions. In contrast, varying pLDDT in the DE loop and terminal regions appear to correlate with different degrees of structural flexibility or disorder. Subsequent structural phylogenetic analysis provided a phylogeny that is in good agreement with previous sequence-based studies. Moreover, the phylogeny supports a parsimonious scenario in which far-red D1 and D1INT evolved from the ancestral form of G4 D1. This study demonstrates the potential of AlphaFold in studies on structural diversity and the evolution of photosynthesis.

bioinformatics↗

bioRxiv2025

Structure of far-red allophycocyanin: stripped down and tuned up for low energy photosynthesis.

A diverse subset of cyanobacteria is capable of transiently modifying their photosynthetic machinery in a process known as far-red light photoacclimation to drive photosynthesis with less energetic photons (700 nm - 800 nm). To achieve this all the main light-driven components of the photosynthetic apparatus, including their allophycocyanin antenna, are replaced with red-shifted paralogues. Recent studies based on the structure of an incomplete complex provided some insights into the tuning of the far-red phycobiliproteins. Here, we solved the structure of the intact bicylindrical allophycocyanin complex from the cyanobacterium Chroococcidiopsis thermalis PCC 7203 at a resolution of 2.61 [A] determined by Cryo-electron microscopy single particle analysis. A comparison between far-red and white light allophycocyanin cores provides insight on the evolutionary adaptations needed to optimize excitation energy transfer in the far-red light adapted photosynthetic apparatus. The reduction in antenna size in far-red photosynthesis, suggests a need to optimize membrane packing to increase the number of photosystems, while the wider spread in the absorption range of the bilins suggests faster and more efficient excitation energy transfer to far-red Photosystem II by limiting backflow of excitation from the reaction centres to the far-red bilin pigments.

biophysics↗

bioRxiv2024

RepeatParam: Algorithm for Parameterising Repeat Proteins and Analysis of Repeat Protein Architectures

MotivationTandem repeat proteins consist of repetitive sequence and structure motifs and have diverse roles in Nature for molecular recognition and signalling. The architecture of repeat proteins can be described using simple helical parameters. Understanding these structural features can inform both the function of these proteins and be used to parametrically design new proteins. Despite their importance, no existing program is capable of completely parameterising repeat proteins. ResultsHere we describe a novel repeat protein parameterisation algorithm, RepeatParam, and a comprehensive repeat protein dataset. RepeatParam determines a helix that defines the global protein architecture and a superhelix that describes the relationship between consecutive repeats. We analyse the relationships between helical parameters and families of different repeat proteins. Availabilityhttps://github.com/dpretorius/repeat-protein-parameterisation

biochemistry↗

bioRxiv2024

Locating the Missing Chlorophylls f in Far-red Photosystem I

The discovery of chlorophyll f-containing oxygenic photosynthesis, with its long-wavelength photochemistry, represented a new low-energy paradigm. However, subsequent structural studies on chlorophyll f-containing Photosystem I (PSI) found five chlorophylls f but none among the photochemically active pigments and concluded that chlorophyll f plays no photochemical role. Here we report a cryo-EM structure (2.01 [A]) of far-red PSI from Chroococcidiopsis thermalis PCC 7203, showing all eight chlorophylls f, including the redox active A-1B. Simulations of absorption difference spectra induced by charge separation indicate that the A-1B chlorophyll f absorbs at 755 nm. The chlorophyll f sites, some wavelength assignments, and conserved far-red-specific amino acids, provide functional insights, including redox tuning of chlorophyll f as the primary donor and far-red excitation energy-sharing over the PSI trimer.

biochemistry↗

bioRxiv2024

SOLeNNoID: A Deep Learning Pipeline For Solenoid Residue Detection in Protein Structures

Solenoid proteins are a subset of tandem repeat proteins, which are structurally distinct from globular proteins. Solenoid proteins are defined by their modular, elongated structures, dependent on interactions between adjacent repeats. These proteins are found across all domains of life and have many important functions such as protein binding, enzymatic catalysis, ice binding and nucleic acid binding. Furthermore, engineered variants of solenoid proteins such as DARPins and designed PPR proteins have therapeutic commercial applications. In order to advance the study of natural solenoid proteins and the design of novel solenoid proteins, accurate tools for solenoid detection and annotation are required. As solenoid structures are more conserved than solenoid sequences and owing to recent developments in protein structure prediction, structure-based solenoid detection is preferred. Here we propose SOLeNNoID - a deep learning pipeline for solenoid residue prediction in protein structures. We cover all three solenoid sub-classes: alpha-, alpha/beta- and beta-solenoids. We use a CNN architecture to reason over protein distance matrices and compare our method to existing structure-based methods. Finally, we produce predictions on the entire PDB and demonstrate a 71 percent increase in solenoid-containing entries over the gold-standard RepeatsDB database using our method. GitHubhttps://github.com/gnik2018/SOLeNNoID ZenodoTBC

bioinformatics↗

bioRxiv2024

Integrated stress response associated with dark microglia promotes microglial lipogenesis and contributes to neurodegeneration

Microglia, the brains primary resident immune cells, are a heterogeneous population and can assume phenotypes with diverse functional outcomes on brain homeostasis. In Alzheimers disease (AD), where microglia are a leading causal cell type, microglia subsets with protective functions have been well characterized. Yet, the identity of microglia subsets that drive neurodegeneration remains unresolved. Here, we identify a neurodegenerative microglia phenotype that is characterized by a conserved stress signaling pathway, the integrated stress response (ISR). Using mouse models to activate or inhibit ISR in microglia, we show that ISR underlies the ultrastructurally distinct "dark" microglia subset linked to pathological synapse loss. Inducing microglial ISR in murine AD models exacerbates neurodegenerative pathologies, such as Tau pathology and synaptic terminal loss. Conversely, inhibiting microglial ISR in AD models ameliorates these pathologies. Mechanistically, we present evidence that ISR promotes the secretion of toxic long-chain lipids that impair neuron and oligodendrocyte homeostasis in vitro. Accordingly, small molecule-based inhibition of lipid synthesis in AD models ameliorates synaptic terminal loss. Our results demonstrate that activation of ISR within microglia represents a novel pathway contributing to neurodegeneration and suggest that this may be sustained, at least in part, by the secretion of long-chain lipids from ISR-activated microglia.

neuroscience↗

bioRxiv2024

The crystal structure of the Shetha Protein II (FeSII) from Azotobacter vinelandii suggests a domain swap

The Azotobacter vinelandii FeSII protein forms an oxygen resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin type ferredoxin, forming a dimer in solution. Previously, the crystal structure was solved (Schlesier et al., 2016), with 5 copies in the asymmetric unit. One copy is a normal adrenodoxin domain, forming a dimer with its crystallographic symmetry mate. The other four copies are in an "open" conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidised and reduced, and the large conformational change in the open configuration allowed binding to nitrogenase. We have independently solved the structure of FeSII in the same crystal form. Our positioning of the atoms in the unit cell is similar to the earlier report. However, our interpretation of the structure is different. We interpret the "open" conformation as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. We caution that crystal structures should be interpreted in terms of the contents of the entire crystal rather than one asymmetric unit.

biochemistry↗

bioRxiv2023

Engraftment and injury repair in regionally conditioned rat lung in vivo by lung progenitors derived from human pluripotent stem cells.

Although lung disease is a major cause of mortality, the mechanisms involved in human lung regeneration are unclear because of the lack of experimental models. Here we report a novel model where human pluripotent stem cell-derived expandable cell lines sharing features of airway secretory and basal cells engraft in the distal rat lung after conditioning by locoregional de-epithelialization followed by irradiation and immunosuppression. The engrafting cells, which we named distal lung epithelial progenitors (DLEPs), contributed to alveolar epithelial cells and generated KRT5-pods, structures involved in distal lung repair after severe injury, but only rarely to distal airways. Most strikingly, however, injury induced by the conditioning regimen was largely prevented by the engrafting DLEPs. The approach described here provides a model to study mechanisms involved in human lung regeneration, and potentially lays the foundation for the preclinical development of cell therapy to treat lung injury and disease.

cell biology↗

bioRxiv2023

DNA damage primes hematopoietic stem cells for direct megakaryopoiesis

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system.1 Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs.2 The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro. Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41+ MK-committed HSCs in vitro. Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenance in vitro. DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.

cell biology↗

bioRxiv2023

Dynamic lid domain of Chloroflexus aurantiacus Malonyl-CoA Reductase controls the reaction

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the split MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.

biochemistry↗

bioRxiv2023

Engraftment of wild-type alveolar type II epithelial cells in surfactant protein C deficient mice

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cellular therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model, Sftpc-/- mice, to provide proof-of-principle for this approach. Sftpc-/- mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to four months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD.

cell biology↗

bioRxiv2022

The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we have used cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the Mn4CaO5 cluster that catalyzes water oxidation, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides new insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

biochemistry↗

bioRxiv2018

Structural basis of light-induced redox regulation in the Calvin cycle

In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.\n\nOne Sentence SummaryHow plants turn off carbon fixation in the dark.

biochemistry↗