Inorg Chem. 2023 Oct 23. doi: 10.1021/acs.inorgchem.3c02620. Online ahead of print.

ABSTRACT

Two organic-inorganic hybrid zinc phosphites incorporating 1,2,4,5-tetrakis(imidazol-1-ylmethyl)benzene (TIMB) molecules were synthesized under hydro(solvo)thermal methods and structurally characterized by single-crystal X-ray diffraction (SCXD). Interestingly, the solvent ratio of water to dimethylformamide induced the formation of a new compound of Zn2(TIMB)0.5(HPO3)2·3H2O (1) and our previously reported structure of Zn2(TIMB)0.5(HPO3)2·H2O (2). Additionally, their dehydrated crystals (1a and 2a) were prepared through heat treatment at 150 °C. SCXD and powder X-ray diffraction showed that all four compounds share the same framework formula of Zn2(TIMB)0.5(HPO3)2 but exhibit a huge difference in their inorganic components and final structures. In 1 and 1a, the inorganic units formed two-dimensional zincophosphite layers, while in 2 and 2a, they formed one-dimensional chains. The inorganic parts of 1 (1a) and 2 (2a) were bridged with TIMB linkers, resulting in 3D structures with rectangular and tubular windows, respectively. Furthermore, 1 was coated on the screen-printed carbon electron as a hybrid material, displaying excellent performance while having a linear relationship with an R2 value of 0.99 within the concentration range of 10-10 to 10-6 mol/L for detecting tryptamine (Try) molecules. Moreover, the results showed that 1 exhibits an ultralow limit of detection of 5.43 × 10-11 mol/L and high specificity toward Try over histamine, ascorbic acid, uric acid, and glucose. The synthesis, structural diversity, stability, and sensing ability are also discussed.

PMID:37870276 | DOI:10.1021/acs.inorgchem.3c02620

EMBO Rep. 2023 Oct 23:e57500. doi: 10.15252/embr.202357500. Online ahead of print.

ABSTRACT

SIRT2, a cytoplasmic member of the Sirtuin family, has important roles in immunity and inflammation. However, its function in regulating the response to DNA virus infection remains elusive. Here, we find that SIRT2 is a unique regulator among the Sirtuin family that negatively modulates the cGAS-STING-signaling pathway. SIRT2 is down-regulated after Herpes simplex virus-1 (HSV-1) infection, and SIRT2 deficiency markedly elevates the expression levels of type I interferon (IFN). SIRT2 inhibits the DNA binding ability and droplet formation of cGAS by interacting with and deacetylating G3BP1 at K257, K276, and K376, leading to the disassembly of the cGAS-G3BP1 complex, which is critical for cGAS activation. Administration of AGK2, a selective SIRT2 inhibitor, protects mice from HSV-1 infection and increases the expression of IFN and IFN-stimulated genes. Our study shows that SIRT2 negatively regulates cGAS activation through G3BP1 deacetylation, suggesting a potential antiviral strategy by modulating SIRT2 activity.

PMID:37870259 | DOI:10.15252/embr.202357500

Elife. 2023 Oct 23;12:e84916. doi: 10.7554/eLife.84916. Online ahead of print.

ABSTRACT

T cells are required to clear infection, moving first in lymph nodes to interact with antigen bearing dendritic cells leading to activation. T cells then move to sites of infection to find and clear infection. T cell motion plays a role in how quickly a T cell finds its target, from initial natiıve T cell activation by a dendritic cell to interaction with target cells in infected tissue. To better understand how different tissue environments might affect T cell motility, we compared multiple features of T cell motion including speed, persistence, turning angle, directionality, and confinement of motion from T cells moving in multiple tissues using tracks collected with microscopy from murine tissues. We quantitatively analyzed natiıve T cell motility within the lymph node and compared motility parameters with activated CD8 T cells moving within the villi of small intestine and lung under different activation conditions. Our motility analysis found that while the speeds and the overall displacement of T cells vary within all tissues analyzed, T cells in all tissues tended to persist at the same speed, particularly if the previous speed is very slow (less than 2 μm/min) or very fast (greater than 8 μm/min) with the exception of T cells in the villi for speeds greater than 10 μm/min. Interestingly, we found that turning angles of T cells in the lung show a marked population of T cells turning at close to 180o, while T cells in lymph nodes and villi do not exhibit this 'reversing' movement. Additionally, T cells in the lung showed significantly decreased meandering ratios and increased confinement compared to T cells in lymph nodes and villi. The combination of these differences in motility patterns led to a decrease in the total volume scanned by T cells in lung compared to T cells in lymph node and villi. These results suggest that the tissue environment in which T cells move can impact the type of motility and ultimately, the efficiency of T cell search for target cells within specialized tissues such as the lung.

PMID:37870221 | DOI:10.7554/eLife.84916

Adv Sci (Weinh). 2023 Oct 23:e2304886. doi: 10.1002/advs.202304886. Online ahead of print.

ABSTRACT

Intravital microscopy (IVM) allows spatial and temporal imaging of different cell types in intact live tissue microenvironments. IVM has played a critical role in understanding cancer biology, invasion, metastases, and drug development. One considerable impediment to the field is the inability to interrogate the tumor microenvironment and its communication cascades during disease progression and therapeutic interventions. Here, a new implantable perfusion window chamber (PWC) is described that allows high-fidelity in vivo microscopy, local administration of stains and drugs, and longitudinal sampling of tumor interstitial fluid. This study shows that the new PWC design allows cyclic multiplexed imaging in vivo, imaging of drug action, and sampling of tumor-shed materials. The PWC will be broadly useful as a novel perturbable in vivo system for deciphering biology in complex microenvironments.

PMID:37870204 | DOI:10.1002/advs.202304886

Cell Genom. 2023 Sep 7;3(10):100402. doi: 10.1016/j.xgen.2023.100402. eCollection 2023 Oct 11.

ABSTRACT

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although clinical risk-defining genomic alterations exist in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography and mutational signatures derived from all variant classes, we identified co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is associated with differences in the mutational processes driving these scenarios, linking risk-defining pathognomonic variants to distinct molecular processes. Whereas high-risk MYCN-amplified neuroblastomas were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas were marked by footprints of chromosome mis-segregation and TOP1 mutational activity. Furthermore, analysis of subclonal mutations uncovered differential activity of these processes through neuroblastoma evolution. Thus, clinical heterogeneity of neuroblastoma patients can be linked to differences in the mutational processes that are active in their tumors.

PMID:37868040 | PMC:PMC10589636 | DOI:10.1016/j.xgen.2023.100402

Cell Genom. 2023 Sep 6;3(10):100401. doi: 10.1016/j.xgen.2023.100401. eCollection 2023 Oct 11.

ABSTRACT

Each human genome has tens of thousands of rare genetic variants; however, identifying impactful rare variants remains a major challenge. We demonstrate how use of personal multi-omics can enable identification of impactful rare variants by using the Multi-Ethnic Study of Atherosclerosis, which included several hundred individuals, with whole-genome sequencing, transcriptomes, methylomes, and proteomes collected across two time points, 10 years apart. We evaluated each multi-omics phenotype's ability to separately and jointly inform functional rare variation. By combining expression and protein data, we observed rare stop variants 62 times and rare frameshift variants 216 times as frequently as controls, compared to 13-27 times as frequently for expression or protein effects alone. We extended a Bayesian hierarchical model, "Watershed," to prioritize specific rare variants underlying multi-omics signals across the regulatory cascade. With this approach, we identified rare variants that exhibited large effect sizes on multiple complex traits including height, schizophrenia, and Alzheimer's disease.

PMID:37868038 | PMC:PMC10589633 | DOI:10.1016/j.xgen.2023.100401

Cell Genom. 2023 Aug 23;3(10):100385. doi: 10.1016/j.xgen.2023.100385. eCollection 2023 Oct 11.

ABSTRACT

Many quantitative trait loci (QTLs) are in non-coding regions. Therefore, QTLs are assumed to affect gene regulation. Gene expression and RNA splicing are primary steps of transcription, so DNA variants changing gene expression (eVariants) or RNA splicing (sVariants) are expected to significantly affect phenotypes. We quantify the contribution of eVariants and sVariants detected from 16 tissues (n = 4,725) to 37 traits of ∼120,000 cattle (average magnitude of genetic correlation between traits = 0.13). Analyzed in Bayesian mixture models, averaged across 37 traits, cis and trans eVariants and sVariants detected from 16 tissues jointly explain 69.2% (SE = 0.5%) of heritability, 44% more than expected from the same number of random variants. This 69.2% includes an average of 24% from trans e-/sVariants (14% more than expected). Averaged across 56 lipidomic traits, multi-tissue cis and trans e-/sVariants also explain 71.5% (SE = 0.3%) of heritability, demonstrating the essential role of proximal and distal regulatory variants in shaping mammalian phenotypes.

PMID:37868035 | PMC:PMC10589627 | DOI:10.1016/j.xgen.2023.100385

iScience. 2023 Oct 4;26(11):108145. doi: 10.1016/j.isci.2023.108145. eCollection 2023 Nov 17.

ABSTRACT

Despite its remarkable potential for transforming low-resolution images, deep learning faces significant challenges in achieving high-quality superresolution microscopy imaging from wide-field (conventional) microscopy. Here, we present X-Microscopy, a computational tool comprising two deep learning subnets, UR-Net-8 and X-Net, which enables STORM-like superresolution microscopy image reconstruction from wide-field images with input-size flexibility. X-Microscopy was trained using samples of various subcellular structures, including cytoskeletal filaments, dot-like, beehive-like, and nanocluster-like structures, to generate prediction models capable of producing images of comparable quality to STORM-like images. In addition to enabling multicolour superresolution image reconstructions, X-Microscopy also facilitates superresolution image reconstruction from different conventional microscopic systems. The capabilities of X-Microscopy offer promising prospects for making superresolution microscopy accessible to a broader range of users, going beyond the confines of well-equipped laboratories.

PMID:37867953 | PMC:PMC10587619 | DOI:10.1016/j.isci.2023.108145

iScience. 2023 Sep 28;26(11):108083. doi: 10.1016/j.isci.2023.108083. eCollection 2023 Nov 17.

ABSTRACT

Bayesian inference is an important method in the life and natural sciences for learning from data. It provides information about parameter and prediction uncertainties. Yet, generating representative samples from the posterior distribution is often computationally challenging. Here, we present an approach that lowers the computational complexity of sample generation for dynamical models with scaling, offset, and noise parameters. The proposed method is based on the marginalization of the posterior distribution. We provide analytical results for a broad class of problems with conjugate priors and show that the method is suitable for a large number of applications. Subsequently, we demonstrate the benefit of the approach for applications from the field of systems biology. We report an improvement up to 50 times in the effective sample size per unit of time. As the scheme is broadly applicable, it will facilitate Bayesian inference in different research fields.

PMID:37867942 | PMC:PMC10589897 | DOI:10.1016/j.isci.2023.108083

iScience. 2023 Sep 30;26(11):108102. doi: 10.1016/j.isci.2023.108102. eCollection 2023 Nov 17.

ABSTRACT

It is widely believed that grid cells provide cues for path integration, with place cells encoding an animal's location and environmental identity. When entering a new environment, these cells remap concurrently, sparking debates about their causal relationship. Using a continuous attractor recurrent neural network, we study spatial cell dynamics in multiple environments. We investigate grid cell remapping as a function of global remapping in place-like units through random resampling of place cell centers. Dimensionality reduction techniques reveal that a subset of cells manifest a persistent torus across environments. Unexpectedly, these toroidal cells resemble band-like cells rather than high grid score units. Subsequent pruning studies reveal that toroidal cells are crucial for path integration while grid cells are not. As we extend the model to operate across many environments, we delineate its generalization boundaries, revealing challenges with modeling many environments in current models.

PMID:37867941 | PMC:PMC10589895 | DOI:10.1016/j.isci.2023.108102

Biol Sport. 2023 Oct;40(4):1169-1176. doi: 10.5114/biolsport.2023.122484. Epub 2023 Apr 6.

ABSTRACT

Different laboratory-based variables are individually associated with cycling performance, but scarce evidence exists on which of them, when all assessed in combination, could best explain cycling performance. The present study aimed to examine the combined association between laboratory-based endurance, strength/power and body composition indicators with time trial performance in high-level cyclists. Ninety-four male cyclists were recruited (age: 20 ± 3.5 years, maximum oxygen uptake [V̇O2max]: 77.7 ± 5.4 ml · kg-1 · min-1). Participants performed a maximal incremental cycling test for the assessment of endurance indicators (peak power output [PPO], V̇O2max, ventilatory threshold [VT] and respiratory compensation point [RCP]), and an incremental loading test to assess muscle strength and power-related outcomes (1-repetition maximum, mean maximal power) in the squat, lunge and hip-thrust exercises. Body composition was assessed by dual energy X-ray absorptiometry. On a separate visit, participants performed a simulated 8-minute time trial to assess cycling performance (determined as the mean power output attained). Strong-to-very-strong correlations were found between all endurance indicators and time trial performance (most r-values ranging between 0.68-0.92), whereas weaker correlations were found for strength/power (r-values < 0.5) or body composition (r-values < 0.7) indicators. Multivariate regression analyses revealed that VT, RCP and PPO explained together 92% of the variance in time trial performance (p < 0.001), with no significant contribution of the remaining variables. Although different endurance, strength/power and body composition individually correlate with simulated time trial performance in high-level cyclists, the former (and particularly VT, RCP and PPO) show the strongest association when all studied in combination. These findings underscore the importance of endurance capabilities (above strength/power or body composition) for maximizing time trial performance.

PMID:37867760 | PMC:PMC10588575 | DOI:10.5114/biolsport.2023.122484

ACS Omega. 2023 Oct 2;8(41):37769-37780. doi: 10.1021/acsomega.3c01663. eCollection 2023 Oct 17.

ABSTRACT

Nanocomposites based on thermoplastic polyurethanes (TPUs) filled with halloysite nanotubes (HNTs) were studied for their physicochemical and biological properties. Nanocomposites containing halloysite nanotube filler contents of 1 and 2% (E+1 and E+2), respectively, were obtained by extrusion. The newly formed E+1 and E+2 nanomaterials exhibited better flexibility and similar thermal properties compared to neat polyurethane. The use of atomic force microscopy (AFM) and differential scanning calorimetry (DSC) thermogram analysis showed that the distribution of halloysite nanotubes in the polymer matrix is more evenly dispersed in the E+1 nanomaterial, where the grains in the E+2 nanomaterial have a greater tendency to form agglomerates. Mechanical tests have shown that nanocomposites with the addition of HNT are characterized by a higher stress at break and elongation at break compared to neat TPU. The results of cytotoxicity tests suggest that the nanocomposite materials express lower toxicity to normal HaCaT and NHDF than to cancer Me45 cells. Further studies showed that the tested materials induced the expression of proinflammatory interleukins IL6 and IL8 in normal cells, but their overexpression in the cancer cell line resulted in cytostatic effects and proliferation reduction. Such a conclusion suggests the possible application of tested materials for regenerative therapies in cancer surgeries.

PMID:37867722 | PMC:PMC10586018 | DOI:10.1021/acsomega.3c01663

ACS Omega. 2023 Oct 2;8(41):38373-38385. doi: 10.1021/acsomega.3c04827. eCollection 2023 Oct 17.

ABSTRACT

The mammalian target of rapamycin (mTOR) is a protein kinase of the PI3K/Akt signaling pathway that regulates cell growth and division and is an attractive target for cancer therapy. Many reports on finding alternative mTOR inhibitors available in a database contain a mixture of active compound data with different mechanisms, which results in an increased complexity for training the machine learning models based on the chemical features of active compounds. In this study, a deep learning model supported by principal component analysis (PCA) and structural methods was used to search for an alternative mTOR inhibitor from mushrooms. The mTORC1 active compound data set from the PubChem database was first filtered for only the compounds resided near the first-generation inhibitors (rapalogs) within the first two PCA coordinates of chemical features. A deep learning model trained by the filtered data set captured the main characteristics of rapalogs and displayed the importance of steroid cores. After that, another layer of virtual screening by molecular docking calculations was performed on ternary complexes of FKBP12-FRB domains and six compound candidates with high "active" probability scores predicted by the deep learning models. Finally, all-atom molecular dynamics simulations and MMPBSA binding energy analysis were performed on two selected candidates in comparison to rapamycin, which confirmed the importance of ring groups and steroid cores for interaction networks. Trihydroxysterol from Lentinus polychrous Lev. was predicted as an interesting candidate due to the small but effective interaction network that facilitated FKBP12-FRB interactions and further stabilized the ternary complex.

PMID:37867669 | PMC:PMC10586184 | DOI:10.1021/acsomega.3c04827

Pest Manag Sci. 2023 Oct 23. doi: 10.1002/ps.7818. Online ahead of print.

ABSTRACT

BACKGROUND: In spite of their importance as arthropod predators, spiders have received little attention in the risk assessment of pesticides. In addition, research has mainly focused on a few species commonly found in agricultural habitats. Spiders living in more natural ecosystems may also be exposed to and affected by pesticides, including insecticides. However, their sensitivity and factors driving possible variation in sensitivity between spider taxa are largely unknown. To fill this gap, we quantified the sensitivity of twenty-eight spider species from a wide range of European ecosystems to lambda-cyhalothrin in an acute exposure scenario.

RESULTS: Sensitivity varied between the tested populations by a factor of 30. Strong differences in sensitivity were observed between families, but also between genera within the Lycosidae. Apart from the variation explained by the phylogeny, spiders from boreal and polar climates were more sensitive than spiders from warmer areas. Overall, the median lethal concentration (LC50 ) of 85% of species was below the recommended application rate of lambda-cyhalothrin (75 ng a.i. cm-2 ).

CONCLUSION: Our study underlines the high sensitivity of spiders to lambda-cyhalothrin, which can lead to unintended negative effects on pest suppression in areas treated with this insecticide. The strong differences observed between families and genera indicate that the functional composition of spider communities would change in affected areas. Overall, the variation in spider sensitivity suggests that multi-species investigations should be more widely considered in pesticide risk assessment. This article is protected by copyright. All rights reserved.

PMID:37867443 | DOI:10.1002/ps.7818

Lancet Microbe. 2023 Oct 19:S2666-5247(23)00213-6. doi: 10.1016/S2666-5247(23)00213-6. Online ahead of print.

ABSTRACT

BACKGROUND: Children with severe acute malnutrition are treated with antibiotics as outpatients. We aimed to determine the effect of 7 days of amoxicillin on acute and long-term changes to the gut microbiome and antibiotic resistome in children treated for severe acute malnutrition.

METHODS: We conducted a secondary analysis of a randomised, double-blinded, placebo-controlled trial (NCT01613547) of amoxicillin in children (aged 6-59 months) with severe acute malnutrition treated as outpatients in Madarounfa, Niger. We randomly selected 161 children from the overall cohort (n=2399) for initial 12-week follow-up from Sept 23, 2013 to Feb 3, 2014. We selected a convenience sample of those 161 children, on the basis of anthropometric measures, for follow-up 2 years later (Sept 28 to Oct 27, 2015). Children provided faecal samples at baseline, week 1, week 4, week 8, week 12, and, for those in the 2-year follow-up cohort, week 104. We conducted metagenomic sequencing followed by microbiome and resistome profiling of faecal samples. 38 children without severe acute malnutrition and six children with severe acute malnutrition matching the baseline ages of the original cohort were used as reference controls.

FINDINGS: In the 12-week follow-up group, amoxicillin led to an immediate decrease in gut microbiome richness from 37·6 species (95% CI 32·6-42·7) and Shannon diversity index (SDI) 2·18 (95% CI 1·97-2·39) at baseline to 27·7 species (95% CI 22·9-32·6) species and SDI 1·55 (95% CI 1·35-1·75) at week 1. Amoxicillin increased gut antibiotic resistance gene abundance to 6044 reads per kilobase million (95% CI 4704-7384) at week 1, up from 4800 (3391-6208) at baseline, which returned to baseline 3 weeks later. 35 children were included in the 2-year follow-up; the amoxicillin-treated children (n=22) had increased number of species in the gut microbiome compared with placebo-treated children (n=13; 60·7 [95% CI 54·7-66·6] vs 36·9 [29·4-44·3]). Amoxicillin-treated children had increased Prevotella spp and decreased Bifidobacterium spp relative to age-matched placebo-treated children, indicating a more mature, adult-like microbiome.

INTERPRETATION: Amoxicillin treatment led to acute but not sustained increases in antimicrobial resistance genes and improved gut microbiome maturation 2 years after severe acute malnutrition treatment.

FUNDING: Bill & Melinda Gates Foundation; Médecins sans Frontières Operational Center Paris; National Institute of Allergy and Infectious Diseases; National Institute of General Medical Sciences; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Edward Mallinckrodt Jr Foundation; Doris Duke Foundation.

PMID:37866373 | DOI:10.1016/S2666-5247(23)00213-6

Placenta. 2023 Oct 17;143:87-90. doi: 10.1016/j.placenta.2023.10.010. Online ahead of print.

ABSTRACT

Trophoblast injury is central to clinically relevant placenta dysfunction. We hypothesized that the mRNA of primary human trophoblasts, exposed to distinct injuries in vitro, capture transcriptome patterns of placental biopsies obtained from common obstetrical syndromes. We deployed a CIBERSORTx deconvolution method to correlate trophoblastic RNAseq-based expression matrices with the transcriptome of omics-defined placental dysfunction patterns in vivo. We found distinct trophoblast injury patterns in placental biopsies from women with fetal growth restriction and a hypertensive disorder, or in biopsies clustered by their omics analysis. Our RNAseq data are useful for defining the contribution of trophoblast injuries to placental dysfunction syndromes.

PMID:37866321 | DOI:10.1016/j.placenta.2023.10.010

Cell J. 2023 Oct 1;25(10):738-740. doi: 10.22074/cellj.2023.2005680.1346.

ABSTRACT

"Theory of Forms" implies that a genuine version of creatures exists beyond the shapes in this world. Stem cell<br />technology has adopted developmental cues to mimic real life. However, the functionality of the lab-made cells is far<br />from primary ones. Perhaps it is time to switch from analytical to systematic perspective in stem cell science. This<br />may be the way to define new horizons based on the systematic perspective and convergence of science in stem cell<br />biology, bridging the current gap between the shadows of real knowledge in current research and reality in future.

PMID:37865882 | DOI:10.22074/cellj.2023.2005680.1346

NPJ Genom Med. 2023 Oct 21;8(1):33. doi: 10.1038/s41525-023-00376-7.

ABSTRACT

Bassoon (BSN) is a component of a hetero-dimeric presynaptic cytomatrix protein that orchestrates neurotransmitter release with Piccolo (PCLO) from glutamatergic neurons throughout the brain. Heterozygous missense variants in BSN have previously been associated with neurodegenerative disorders in humans. We performed an exome-wide association analysis of ultra-rare variants in about 140,000 unrelated individuals from the UK Biobank to search for new genes associated with obesity. We found that rare heterozygous predicted loss of function (pLoF) variants in BSN are associated with higher BMI with p-value of 3.6e-12 in the UK biobank cohort. Additionally, we identified two individuals (one of whom has a de novo variant) with a heterozygous pLoF variant in a cohort of early onset or extreme obesity and report the clinical histories of these individuals with non-syndromic obesity with no history of neurobehavioral or cognitive disability. The BMI association was replicated in the All of Us whole genome sequencing data. Heterozygous pLoF BSN variants constitute a new etiology for obesity.

PMID:37865656 | DOI:10.1038/s41525-023-00376-7

Trends Cell Biol. 2023 Oct 19:S0962-8924(23)00197-6. doi: 10.1016/j.tcb.2023.09.005. Online ahead of print.

ABSTRACT

The rising prevalence of kidney diseases urges the need for novel therapies. Kidney organoids and tubuloids are advanced in vitro models and have recently been described as promising tools to study kidney (patho)physiology. Recent developments have shown their application in disease modeling, drug screening, and nephrotoxicity. These applications rely on their ability to mimic (dys)function in vitro including endocrine activity and drug, electrolyte, and water transport. This review provides an overview of these emerging kidney models and focuses on the most recent developments that utilize their functional capabilities. In addition, we cover current limitations and provide future perspectives for this rapidly evolving field, including what these functional properties mean for translational and personalized medicine now and in the future.

PMID:37865608 | DOI:10.1016/j.tcb.2023.09.005

J Biol Chem. 2023 Oct 19:105371. doi: 10.1016/j.jbc.2023.105371. Online ahead of print.

ABSTRACT

Ca2+/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in regulation of cellular processes in a variety of tissues including brain, heart, liver and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phoshoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs (gRNAs) targeting the CAMK2D catalytic domain to create multiple CAMK2D knock-out cell lines. AQP2 protein abundance was lower in the CAMK2D knockout cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D knockout clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro phosphorylation studies using recombinant CAMK2D. Thirty-five of the significantly down-regulated phosphorylation sites in CAMK2D knock-out cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.

PMID:37865316 | DOI:10.1016/j.jbc.2023.105371