Biology subjects

Buck, J.

Publications and source records attributed to Buck, J..

11 recordsLinked to original sources

bioRxiv2025

CD8 T cells mediate immunosurveillance for neoantigen+ epithelial stem cells in the colon

Epithelial cells in the colon accumulate substantial numbers of somatic mutations, some of which can be recognised as neoantigens. The ability of CD8 T cells to survey for neoantigen+ cells in the healthy colon would provide an early detection mechanism to prevent cancer, but it is unclear whether neoantigen-specific CD8 T cells can mediate this process of immunosurveillance without becoming tolerant. To address this question, we used a genetically engineered mouse model to express a neoantigen in the epithelial cells of the adult proximal colon. Induction of neoantigen expression led to rapid elimination of neoantigen+ epithelial cells from the colon in a CD8 T cell-dependent manner. Neoantigen-specific CD8 T cells acquired cytolytic function within the colon tissue under steady-state conditions, which was required for elimination of the neoantigen+ epithelial cells. Despite the elimination of [~]25% of their epithelial cells over a two-day period, the colons looked histologically normal. Immunofluorescence and single-cell transcriptomic analyses revealed that neoantigen-specific CD8+ T cells specifically target neoantigen+ stem cells at the crypt base, which was associated with Ki67 in the crypt wall and abundance of neoantigen-negative stem cells. Infiltrating neoantigen-specific CD8 T cells made IFNg and expressed PD-1, raising the question of why PD-1-dependent suppression did not prevent the acquisition of effector functions by these neoantigen-specific CD8 T cells. Despite an increased signature of interferon-stimulated genes in colonic epithelial cells, PD-L1 expression was surprisingly absent. Moreover, we found that colonic epithelial stem cells also did not express PD-L1 under conditions of chronic inflammation, such as ulcerative colitis, immune checkpoint-induced colitis, and ageing, or when directly stimulated with IFN-{gamma} in vitro. Analyses of the PD-L1 gene promoter across humans and mice showed hypermethylation at sites associated with PD-L1 repression in cancer. Thus, our data support a model in which the acquisition of neoantigens by colonic epithelial cells triggers CD8 T cell-mediated immunosurveillance. This results in the elimination of PD-L1-negative neoantigen+ stem cells by effector CD8 T cells and simultaneous repair of the colon by neoantigen-negative epithelial cells to prevent immunopathology.

immunology↗

bioRxiv2025

Sperm meet the elevated energy demands to attain fertilization competence by increasing flux through aldolase

Prior to ejaculation, sperm are stored in the epididymis in a resting metabolic state. Upon ejaculation, sperm must alter their metabolism to generate the energy needed to support the motility and maturation process known as capacitation to reach and fertilize the oocyte. How sperm regulate the capacitation-induced increase in carbon flux is unknown. Here, we use 13C stable isotope labeling to follow glucose metabolism through sperm central carbon metabolic network before and after sperm activation. We identify regulatory steps which sperm use to alter their metabolic state from resting to highly active. In activated sperm, glucose flux through glycolysis is increased at the expense of the pentose phosphate pathway to increase energy yield. Increased glycolytic activity seems to be due to capacitation-induced stimulation of flux through aldolase. In the mitochondria-containing midpiece, glycolytically generated pyruvate feeds the TCA cycle to further maximize energy yield via oxidative phosphorylation. In the mitochondria-free principal piece of the tail, pyruvate produced from glycolysis is reduced to lactate by lactate dehydrogenase. Reduction to lactate regenerates oxidized NAD+ ensuring a sufficient supply to support glycolysis. The resultant lactate is at least partially secreted. Finally, we find evidence that there is an as yet unknown endogenous source of energy in sperm feeding the upregulation of TCA cycle intermediates. These studies provide the most complete picture of the metabolic shift which occurs in capacitating sperm. Significance statementA rapid switch from a quiescent to a high energy-demanding state during ejaculation is essential for sperm to reach and fertilize the oocyte. Somatic cells also undergo bioenergetic switches from low to very high energy demand. However, because metabolic processes essential for proliferation are going on in parallel, it is difficult to identify the molecular mechanisms regulating the increase in ATP production. This study represents the first complete picture of the metabolic reprogramming that happens in sperm upon ejaculation. Using stable isotope labeling, we identify rate-limiting enzymatic steps and points of regulation directing the changes in metabolic flux. Our sperm metabolic studies allow us to identify conserved mechanisms of metabolic regulation that are crucial for the survival of mammalian cells.

biochemistry↗

bioRxiv2025

A corticostriatal learning mechanism linking excess striatal dopamine and auditory hallucinations

1Auditory hallucinations are linked to elevated striatal dopamine, but their underlying computational mechanisms have been obscured by regional heterogeneity in striatal dopamine signaling. To address this, we developed a normative circuit model in which corticostriatal plasticity in the ventral striatum is modulated by reward prediction errors to drive reinforcement learning while that in the sensory-dorsal striatum is modulated by sensory prediction errors derived from internal belief to drive self-supervised learning. We then validate the key predictions of this model using dopamine recordings across striatal regions in mice, as well as human behavior across various tasks including a new hybrid learning task. Finally, we find that changes in learning resulting from optogenetic stimulation of the sensory-dorsal striatum in mice and individual variability in hallucinations in humans are best explained by selectively enhancing dopamine levels in the model sensory-dorsal striatum. These findings identify plasticity mechanisms underlying biased learning of sensory expectations as a biologically plausible link between excess dopamine and hallucinations.

neuroscience↗

bioRxiv2025

X-ray Spectroscopy Meets Native Mass Spectrometry: Probing Gas-phase Protein Complexes

Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.

biochemistry↗

bioRxiv2024

ColBuilder: Flexible structure generation of crosslinked collagen fibrils

Collagen fibrils are fundamental building blocks of connective tissues, yet generating accurate molecular models of their structure remains challenging due to their hierarchical organization and complex crosslinking patterns. ColBuilder has been developed to automate the generation of atomistic models of crosslinked collagen fibrils and facilitate the setup of molecular simulations. The tool integrates homology modeling, higher-order structure generation and optimization to build complete fibril structures with precise control over sequence composition, crosslinking patterns, and dimensions. Users can explore different collagen sequences, manipulate crosslink chemistry through mixed ratios and densities, and generate fibrils of varying diameter and length. All-atom molecular dynamics simulations of 335 nm-long fibrils validate the generated structures, showing excellent agreement with experimental measurements of D-band periodicity and force-extension behavior. ColBuilder is available both as an open-source command-line application and through a web interface at colbuilder.mpip-mainz.mpg.de.

biophysics↗

bioRxiv2023

Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αvβ3-targeted iron oxide microparticles

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly anti-angiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a peptide (RGD) targeting the integrin v{beta}3. The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastases in vivo. A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T2* weighted imaging was used to visualize MPIO induced hypointense voxels in vivo, and Prussian blue staining was used to visualize MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualizing angiogenic vessels per se was reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. Whilst pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.

cancer biology↗

bioRxiv2022

Amyloidogenic Propensity of Self-Assembling Peptides and their Adjuvant Potential for use as DNA Vaccines

De novo designed peptides that self-assemble into cross-{beta} rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-{beta} peptide KFE8, compared to pathological amyloid and amyloid-like proteins -synuclein (-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a fibrillizing scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and can be efficiently cleared by autophagy. We also demonstrate that synthetic KFE8 nanofibers do not cross-seed amyloid formation of -syn in mammalian cells compared to -syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP or -syn tagged GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-{beta}-rich peptide nanofibers have distinct properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles.

bioengineering↗

bioRxiv2021

External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis

The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea. Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2 {leftrightarrow} HCO3- (bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

biophysics↗

bioRxiv2021

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

Soluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperms journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

pharmacology and toxicology↗

bioRxiv2020

Non-canonical regulation of glycogenolysis and the Warburg phenotype by soluble adenylyl cyclase

Cyclic AMP is produced in cells by two very different types of adenylyl cyclases: the canonical transmembrane adenylyl cyclases (tmACs, ADCY1[~]9) and the evolutionarily more conserved soluble adenylyl cyclase (sAC, ADCY10). While the role and regulation of tmACs is well documented, much less is known of sAC in cellular metabolism. We demonstrate here that sAC is an acute regulator of glycolysis, oxidative phosphorylation and glycogen metabolism, tuning their relative bioenergetic contributions. Suppression of sAC activity leads to aerobic glycolysis, enhanced glycogenolysis, decreased oxidative phosphorylation, and an elevated cytosolic NADH/NAD+ ratio, resembling the Warburg phenotype. Importantly, we found that glycogen metabolism is regulated in opposite directions by cAMP depending on its location of synthesis and downstream effectors. While the canonical tmAC-cAMP-PKA axis promotes glycogenolysis, we identify a novel sAC-cAMP-Epac1 axis that suppresses glycogenolysis. These data suggest that sAC is an autonomous bioenergetic sensor that suppresses aerobic glycolysis and glycogenolysis when ATP levels suffice. When the ATP level falls, diminished sAC activity induces glycogenolysis and aerobic glycolysis to maintain energy homeostasis.

biochemistry↗

bioRxiv2018

Integrative analysis of Paneth cell proteomic data from intestinal organoids reveals functional processes affected in Crohn’s disease due to autophagy impairment

Summary statementUsing an integrative approach encompassing intestinal organoid culture, proteomics, transcriptomics and protein-protein interaction networks, we list Paneth cell functions dependent on autophagy.\n\nAbstractPaneth cells are key epithelial cells providing an antimicrobial barrier and maintaining integrity of the small intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and often associated with digestive pathologies such as Crohns disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process which recycles cellular components. The direct effect of autophagy-impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells making these cells impaired in autophagy. Using 3D intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type (WT) and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy targeted proteins and functional information to identify the mechanistic link between autophagy-impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%), predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms degrading proteins key to Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration affect protein level and not gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred down-regulation of exocytosis. Our observations could explain how protein level alterations affect Paneth cell homeostatic functions upon autophagy impairment.

systems biology↗