Biology subjects

Messinger, J.

Publications and source records attributed to Messinger, J..

7 recordsLinked to original sources

bioRxiv2025

Atomic resolution structure of spinach rubisco reveals protons and dynamics

Photosynthetic organisms sustain life on Earth by storing solar energy in biomass. Central to this process is rubisco, the enzyme that catalyses the fixation of CO2 to ribulose-1,5-bisphosphate, providing the primary gateway for inorganic carbon into the biosphere. Rubiscos catalytic efficiency is a major determinant of crop productivity and global carbon flux, making it a longstanding target for protein engineering.1-4 Yet, attempts to enhance its performance through rational design have met limited success due to an incomplete understanding of rubiscos catalytic mechanism. Here, we report an atomic resolution (1.25 [A]) cryo-EM structure of spinach rubisco in complex with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate. Supported by large-scale quantum/classical (QM/MM) calculations, our structural analysis reveals protonation equilibria within the active site and unexpected structural flexibility across large protein regions despite the exceptionally tight ligand binding. Our findings provide new insight into the complex interplay of protonation equilibria and conformational sampling, suggesting a novel basis for rubiscos rational redesign utilizing strategies that rely on a combination of dynamic and electrostatic control.

biochemistry↗

bioRxiv2025

Selective Knockout of Murine Glutamic Acid-rich Protein 2 (GARP2) Significantly Alters Cellular Dark Noise in Rod Photoreceptors

GARP2, a glutamic-acid-rich protein found exclusively in rod photoreceptors, has been suggested to function as a structural protein, a modulator of the cGMP phosphodiesterase enzyme (PDE6), and a gating inhibitor of the rod cGMP-gated cation channel. GARP2 is a splice variant of the Cngb1 gene, which in the rods encodes the {beta}-subunit of the cyclic nucleotide-gated cation channel. Mutations in Cngb1 cause retinitis pigmentosa (RP45), and {beta}-subunit knockout mice are being studied as models of this disease. In this study, using ZFN-mediated gene editing, we have selectively eliminated GARP2 expression, while not affecting expression of the cyclic nucleotide gated cation channel {beta} -subunit, to determine its essential functions in mouse rods. The absence of GARP2 caused no consistent perturbations of retinal structure. Transiently, rod outer segment length was regionally greater than WT and infrequently misaligned, appearing parallel to the retinal pigment epithelium. Electroretinography of the knockout mice did reveal consistent functional alterations over time, seen as a reduction in the ERG response amplitudes in older mice, albeit with no significant alterations in sensitivity to light. Interestingly, single-cell patch-clamp recordings showed a significant reduction in rod photoreceptor dark noise consistent with a previously proposed role for GARP2 in binding to PDE6 and affecting its basal activity. Our results suggest a role for the GARP2-PDE6 interaction in stabilizing the PDE6 enzyme and controlling the turnover rate of cGMP in darkness, adjusting the level of dark noise and implicating an influence on the signal and noise properties of rod photoreceptors. Key PointsO_LIGlutamic acid-rich protein 2 (GARP2), an alternatively spliced variant of the Cngb1 gene, is exclusively expressed in rod photoreceptors, but the in vivo role of GARP2 remains unestablished. C_LIO_LIWe used precision gene editing technology to selectively knockout soluble GARP2 expression in rods, to determine its essential functions in structure and function of the retina. C_LIO_LIWe show that GARP2 has a minor role in maintaining the structural integrity of the rod outer segments as a function of age. C_LIO_LIOur study indicates that GARP2 has an important role in regulating rod photoreceptor dark noise, likely through stabilization of PDE6 basal activity to maintain the appropriate cGMP turnover rate. C_LIO_LIThis regulation is critical to facilitate the single-photon sensitivity and function of the rod photoreceptors. C_LI

neuroscience↗

bioRxiv2025

Modeling Retinitis Pigmentosa 59: Dhdds T206A and Dhdds K42E knock-in mutant mice are phenotypically similar.

Dehydrodolichyl diphosphate synthase (DHDDS) is an essential enzyme required for several forms of protein glycosylation in all eukaryotic cells. Surprisingly, three mutant alleles, (DhddsK42E/K42E (K42E/K42E), DhddsT206A/K42E (T206A/K42E), and found in only one patient, DhddsR98W/K42E (R98W/K42E) have been reported that cause non-syndromic retinitis pigmentosa (RP59), an inherited retinal degeneration (IRD). Because T206A was only observed heterozygously with the K42E allele in RP59 patients, we used CRISPR/CAS9 technology to generate T206A/T206A, and subsequently T206A/K42E alleles in mice to assess the contribution of the T206A allele to the disease phenotype, to model the human disease, and to compare resulting phenotypes to our homozygous K42E mouse model. By postnatal (PN) 12-mo, T206A/K42E mice exhibit significant reduction of inner nuclear layer thickness as was observed in K42E/K42E mice. No change in outer nuclear layer thickness is observed in all mutant phenotypes up to PN 12 mo. Electroretinography (ERG) showed a significantly reduced b-wave without a-wave decrement and by PN 3-mo, ERG c- and d-wave responses were significantly attenuated in all phenotypes. Consistent with a reduction in inner nuclear layer thickness seen by OCT and cell loss observed by histology, bipolar and amacrine cell densities were reduced in all Dhdds mutant phenotypes compared to PN 8-12 mo age-matched controls. These results indicate that the DHDDS T206A allele causes retinal disease independent of the K42E allele, and that there likely is a common disease mechanism involving RP59-associated DHDDS mutations. We conclude that the physiological basis of retinal dysfunction in RP59 involves defective signaling in the inner retina resulting in bipolar/amacrine cell degeneration.

cell biology↗

bioRxiv2024

Cryo-EM insight into hydrogen positions and water networks in photosystem II

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thereby sustains life on Earth. It catalyzes two chemical reactions, plastoquinone reduction and water oxidation to molecular oxygen, which both are performed at sequestered sites. While it is known that proton-coupled electron transfer is crucial for these processes, the molecular details have remained speculative due to incomplete structural data. Thus, we collected high-resolution cryo-EM data of photosystem II from Thermosynechococcus vestitus. The advanced structure (1.71 [A]) reveals several previously unditected occupied water binding sites and more than half of the hydrogen and proton positions of the protein. This unprecedented insight into the structure of photosystem II significantly enhances our understanding of its intricate protein-water-cofactor interactions enabling solar-driven catalysis. One sentence summaryCryo-EM structure of PSII at 1.71 [A] resolution reveals over 50% of hydrogen and proton sites and additional water binding sites, aiding catalytic insight.

biochemistry↗

bioRxiv2022

O2 photoreduction at acceptor side of Photosystem I provide photoprotection to conifer thylakoids in early spring

Green organisms evolve O2 via photosynthesis and consume by respiration. Net O2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles demonstrate strong O2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring. This phenomenon deviates from the general finding that photosynthetic organisms evolve O2 upon illumination. By using different electron transport chain inhibitors, we showed that O2 consumption occurred around photosystem (PS) I and correlated with higher abundance of flavodiiron (Flv) A protein in ES thylakoid membranes. Furthermore, by measuring P700 absorption changes, we separated different alternative electron flow pathways and demonstrated that electron scavenging from the acceptor-side of PSI via O2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

plant biology↗

bioRxiv2019

Integrative single-cell and bulk RNA-seq analysis in human retina identified cell type-specific composition and gene expression changes for age-related macular degeneration

Age-related macular degeneration (AMD) preferentially affects distinct cell types and topographic regions in retina. To characterize the impact of AMD on gene expression changes across retinal cell types and regions, we generated both single-cell RNA-seq (scRNA-seq) and bulk RNA-seq data from macular and peripheral retina in postmortem human donors with and without AMD. The scRNA-seq data revealed 11 major cell types with many previously reported AMD risk genes showing substantial cell type and region specificity. Cell type proportional changes with advancing AMD stage were significant for Muller glia, rods, astrocytes, microglia and endothelium.

genomics↗

bioRxiv2018

Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

Ventral tegmental area (VTA) dopamine (DA) neurons perform diverse functions in motivation and cognition, but their precise roles in addiction-related behaviors are still debated. Here, we targeted VTA DA neurons for bidirectional chemogenetic modulation during specific tests of cocaine reinforcement, demand, and relapse-related behaviors, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of Gq-, but not Gs-signaling in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory Gi/o signaling in DA neurons blunted cocaines reinforcing and priming effects, reduced stress-potentiated reinstatement, and altered cue-induced cocaine seeking strategy, but not the motivational impact of cocaine cues per se. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein signaling, and DA neuron efferent target, highlighting their multifaceted roles in addiction.\n\nSignificance StatementG-protein coupled receptors are crucial modulators of VTA dopamine neuron activity, but how metabotropic signaling impacts dopamines complex roles in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs during a variety of cocaine seeking behaviors, revealing nuanced, pathway-specific roles in cocaine reward, effortful seeking, and relapse-like behaviors. Gq- and Gs-stimulation activated dopamine neurons, but only Gq stimulation robustly enhanced cocaine seeking. Gi/o inhibitory signaling altered the response strategy employed during cued reinstatement, and reduced some, but not all types of cocaine seeking. Results show that VTA dopamine neurons modulate numerous distinct aspects of cocaine addiction- and relapse-related behaviors, and indicate potential new approaches for intervening in these processes to treat addiction.

neuroscience↗