Biology subjects

Kang, J.

Publications and source records attributed to Kang, J..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction

Excessive alcohol consumption poses significant health risks and is closely associated with oxidative damage. The KEAP1-NRF2-ARE signaling pathway serves as the primary antioxidant system. However, current small molecule inhibitors are all covalently bound to KEAP1, meaning that once bound, they are not easily dissociated, while continuous inhibition of KEAP1 exhibits severe side effects. In this study, BLI, CETSA, Pull-down, Co-IP and HDX-MS assay analysis were conducted to detect the KEAP1 binding behavior of natural product, capsaicin (CAP), both in vitro and in cells. The ethanol-induced acute gastric mucosal damage rat model was also established to evaluate the therapeutic effect of CAP. Our findings demonstrated that CAP mitigated mitochondrial damage, facilitated the nuclear translocation of NRF2, leading to the up-regulation of downstream antioxidant response elements, HMOX1, TXN, GSS and NQO1 in GES-1 cells. Furthermore, CAP directly bind to KEAP1 and inhibit the interaction between KEAP1 and NRF2. In the KEAP1-knockout 293T cells, CAP failed to activate NRF2 expression. We identified that CAP non-covalently bound to Kelch domain and allosterically regulated three specific regions of KEAP1 : L342-L355, D394-G423 and N482-N495. To improve drug solubility and delivery efficiency, we developed IR-Dye800 modified albumin coated CAP nanoparticles. The nanoparticles significantly reduced the gastric mucosal inflammation and activated NRF2 downstream genes in vivo. Our hypothesis was further verified our hypothesis in Nfe2l2-knockout mice. This study provides new insights that CAP is a safe and novel NRF2 agonist by allosterically regulating KEAP1, which may contribute to the development of lead drugs for oxidative stress-related illness, e.g. aging, cancer, neurodegenerative and cardiovascular diseases.

cell biology↗

bioRxiv2023

Identifying drug sensitivity of multifocal primary prostate cancer

The pronounced intra-patient variability in multifocal primary prostate cancer (PCa) has curtailed the efficacy of current treatment options. Patient-derived organoids (PDOs) have emerged as a pivotal model for functional testing due to their capability to retain the histopathological and molecular characteristics of parental tissues, allowing timely acquisition of drug response outcomes. In our study, employing twin biopsies from multiple lesions with matched PDO models in vitro, we investigated the molecular heterogeneity of PCa, and how it is linked to in vitro PDO pharmacological heterogeneity. Our functional testing approach leverages PDOs to screen standard-of-care treatment and FDA-approved compounds for other malignancies, aiming to repurpose their use in PCa and explore alternatives to androgen deprivation therapy. By integrating gene expression data from parental tissue with drug response results from PDOs, we have established a transcriptomics-based drug prediction models. The machine learning-based prediction model can predict the experimental PDO response to a specific drug, for the majority of screened drugs. This study offers a preclinical approach to potentially procure drug prediction outcomes and validate them in PDO models, as a prior step to clinical trial investigations or for selection of targeted therapeutic options.

cancer biology↗

bioRxiv2023

Cell autonomous role of leucine-rich repeat kinase in protection of dopaminergic neuron survival

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinsons disease (PD), which is the leading neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). However, whether LRRK2 mutations cause PD and degeneration of DA neurons via a toxic gain-of-function or a loss-of-function mechanism is unresolved and has pivotal implications for LRRK2-based PD therapies. In this study, we investigate whether LRRK2 and its functional homologue LRRK1 play an essential, intrinsic role in DA neuron survival through the development of DA neuron-specific LRRK conditional double knockout (cDKO) mice. We first generated and characterized floxed LRRK1 and LRRK2 mice and then confirmed that germline deletions of the floxed LRRK1 and LRRK2 alleles result in null mutations, as evidenced by the absence of LRRK1 and LRRK2 mRNA and protein in the respective homozygous deleted mutant mice. We further examined the specificity of Cre-mediated recombination driven by the dopamine transporter-Cre (DAT-Cre) knockin (KI) allele using a GFP reporter line and confirmed that DAT-Cre-mediated recombination is restricted to DA neurons in the SNpc. Crossing these validated floxed LRRK1 and LRRK2 mice with DAT-Cre KI mice, we then generated DA neuron-restricted LRRK cDKO mice and further showed that levels of LRRK1 and LRRK2 are reduced in dissected ventral midbrains of LRRK cDKO mice. While DA neuron-restricted LRRK cDKO mice of both sexes exhibit normal mortality and body weight, they develop age-dependent loss of DA neurons in the SNpc, as demonstrated by the progressive reduction of DA neurons in the SNpc of LRRK cDKO mice at the ages of 20 and 24 months but the unaffected number of DA neurons at the age of 15 months. Moreover, DA neurodegeneration is accompanied with increases of apoptosis and elevated microgliosis in the SNpc as well as decreases of DA terminals in the striatum, and is preceded by impaired motor coordination. Taken together, these findings provide the unequivocal evidence for the importance of LRRK in DA neurons and raise the possibility that LRRK2 mutations may impair its protection of DA neurons, leading to DA neurodegeneration in PD.

neuroscience↗

bioRxiv2023

A robust knock-in approach using a minimal promoter and a minicircle

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.

developmental biology↗

bioRxiv2023

ATP energy-independentlymodulates the folding equilibrium of ALScausing C71G-hPFN1

C71G is the most aggregation-prone and toxic mutant of 140-residue human profin-1 (hPFN1) that causes familial ALS by gain of toxicity, but its underlying mechanisms still remain unknown. C71G-hPFN1 exists in an equilibrium between folded and unfolded states, whose dynamic/thermodynamic properties and modulation are not yet defined. Here, we utilized NMR to quantify the populations to be 55.2% and 44.8% respectively for folded and unfolded states exchanging at 11.7 Hz. Intriguingly, even the folded state of C71G-hPFN1 has increased ps-ns flexibility and reduced thermodynamic stability, thus rationalizing its high aggregation-proneness. Strikingly, C71G-hPFN1 provides a unique model to unambiguously visualize the effects of ATP and 11 related molecules on its folding equilibrium by NMR. Unexpectedly, ATP completely converted C71G-hPFN1 into the folded state at 1:2, which is physiologically relevant in most living cells. By contrast, TMAO, a well-known protein-folding inducer, showed no detectable conversion even at 1:2000. Surprisingly, the inducing capacity of ATP comes from its triphosphate group, but free triphosphate strongly triggered aggregation. The inducing capacity was determined to rank as: ATP = ATPP = PPP > ADP = AMP-PNP = AMP-PCP = PP, while AMP, Adenosine, P and NaCl showed no detectable capacity. Mechanistically, ATP and triphosphate appear to enhance the intrinsic folding capacity encoded by the sequence. Therefore, by joining Adenosine and triphosphate ATP appears to integrate three abilities: inducing folding, inhibiting aggregation and increasing stability. Our study provide a mechanism for the finding that some single-cell organisms still use polyphosphates as primordial chaperones. Moreover, ATP continue to play foundational roles in modern cells, shedding light on the longstanding enigma of the age-related onset of FALS, which coincides with the ageing-dependent reductions in ATP concentrations.

biophysics↗

bioRxiv2023

Combined representation of visual features in the scene-selective cortex

Visual features of separable dimensions like color and shape conjoin to represent an integrated entity. We investigated how visual features bind to form a complex visual scene. Specifically, we focused on features important for visually guided navigation: direction and distance. Previously, separate works have shown that directions and distances of navigable paths are coded in the occipital place area (OPA). Using functional magnetic resonance imaging (fMRI), we tested how separate features are concurrently represented in the OPA. Participants saw eight different types of scenes, in which four of them had one path and the other four had two paths. In single-path scenes, path direction was either to the left or to the right. In double-path scenes, both directions were present. Each path contained a glass wall located either near or far, changing the navigational distance. To test how the OPA represents paths in terms of direction and distance features, we took three approaches. First, the independent-features approach examined whether the OPA codes directions and distances independently in single-path scenes. Second, the integrated-features approach explored how directions and distances are integrated into path units, as compared to pooled features, using double-path scenes. Finally, the integrated-paths approach asked how separate paths are combined into a scene. Using multi-voxel pattern similarity analysis, we found that the OPAs representations of single-path scenes were similar to other single-path scenes of either the same direction or the same distance. Representations of double-path scenes were similar to the combination of two constituent single-paths, as a combined unit of direction and distance rather than pooled representation of all features. These results show that the OPA combines the two features to form path units, which are then used to build multiple-path scenes. Altogether, these results suggest that visually guided navigation may be supported by the OPA that automatically and efficiently combines multiple features relevant for navigation and represent a navigation file.

neuroscience↗

bioRxiv2023

Anti-muscle Atrophy Effects of Lactobacillus reuteri ATG-F4 and Its Impact on Intestinal Microbiota and Metabolites: Implications for Prophylaxis and Therapy

Lactobacillus reuteri ATG-F4, human gut-derived bacteria, was orally administrated in a model of hindlimb immobilization and confirmed the muscular performance, muscle mass and mechanism on anti-atrophy study. Concomitantly, the changes in the intestinal flora, the metabolites and cytokines were investigated. In the stapled immobilization mice model, ATG- F4 treated group had significantly increased muscle mass, myofiber size, running time to be exhausted and grip strength. The cytokine levels in serum and muscle tissues were reduced by ATG-F4 treatment. Furthermore, the phosphorylation of proteins involved in muscle synthesis such as mTOR, p70S6K, rpS6 and 4E-BP1 increased and MuRF1 related to muscle atrophy factor reduced in the TA and GA muscles. ATG-F4 treatment changed the ratio of main intestinal microflora by increasing the family Muribaculaceae (phylum Bacteroidetes) and decreasing the family Lachnospiraceae (phylum Firmicutes) and Lactobacillaceae (phylum Firmicutes). Also, the level of short chain fatty acids (SCFAs) including butyric acid and acetic acid in the serum of ATG-F4 group were increased. These results suggest that L. reuteri ATG- F4 can inhibit muscle atrophy and it is associated with the microbiota and its metabolites with the anti-inflammation effect. ATG-F4 may be a potential prophylactic or therapeutic composition for muscle atrophy.

physiology↗

bioRxiv2023

Optimal Transport Method-Based Gene Filter (GF) Denoising Algorithm for Enhancing Spatially Resolved Transcriptomics Data

The recent advancements in spatially resolved transcriptomics (SRT) technology have enabled the acquisition of gene expression data at near- or sub-single-cell resolution, along with simultaneous imaging of physical locations. Nevertheless, necessary experimental procedures such as tissue fixation, permeabilization, and tissue removal inevitably induce the diffusion of transcribed molecules. Consequently, this leads to the partial capture of ex-situ transcripts in SRT data, thereby introducing a considerable amount of noise into the dataset. To address this issue, in this study, we focused on evaluating the diffusion pattern of individual genes within tissue regions and quantitatively calculating their signal-to-noise ratio (SNR). Through this analysis, we successfully identified "invalid genes" exhibiting widespread expression across tissue regions. Then by filtering out these genes, we effectively reduced the high noise level present in SRT data. To achieve this, we developed the gene filter denoising (GF) algorithm, which utilizes the optimal transport method to compute the gene diffusion coefficient and generate denoised SRT data. One notable advantage of our GF algorithm is its ability to fully "respect" the raw sequencing data, thereby avoiding the introduction of false positives often associated with traditional interpolation and modification denoising methods. Furthermore, we conducted comprehensive validation of GF, and the GF-denoised SRT data demonstrated substantial improvements in clustering, identification of differentially expressed genes (DEGs), and cell type annotation. Taken together, we believe that the GF denoising technique will serve as an essential and crucial step in exploring SRT data and investigating the underlying biological processes.

bioinformatics↗

bioRxiv2023

Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

Resident macrophages and infiltrating monocytes in kidneys of patients with lupus nephritis are altered both in frequency and function relative to their counterparts in healthy kidneys. The extent to which mouse models might be useful in developing approaches to target these cells for treating lupus nephritis is poorly understood. Here, we studied four common lupus mouse models that share clinical, serologic, and histopathologic kidney changes with humans. Using single-cell profiling and multiplex spatial imaging to analyze the intrarenal myeloid compartment with the onset of clinical disease in these models, we identified monocyte and macrophage subsets that expand or contract in kidneys with clinical nephritis. A unique subset of classical monocytes expanded with the onset of disease and expressed genes such as CD9, Spp1, Ctsd, Cd63, Apoe, and Trem2 that were previously shown to be induced by tissue injury and play a role in inflammation, lipid metabolism and tissue repair in other organs. Resident macrophages transitioned from a pro-inflammatory to a similar injury-associated state with onset of disease. To test whether these findings in mouse models were also observed in humans, we re-analyzed monocytes and macrophages in a single-cell RNAseq dataset of kidney biopsies from 155 patients with lupus nephritis and 30 healthy donors, collected by the NIH AMP RA/SLE consortium. Human monocytes and macrophages showed conserved changes in gene expression programs associated with lupus nephritis disease indices, and localized to similar kidney microenvironments as in mice. By identifying myeloid subsets and disease-associated alterations in biological processes that are conserved across species, we provide a strong rationale for functional studies of these cells and pathways in mice to uncover mechanisms and find targets relevant to human lupus nephritis. One sentence summaryThis study characterizes intrarenal myeloid cells from four lupus mouse models and 155 patients with lupus nephritis using single-cell RNA-seq and imaging, and identifies novel infiltrating and resident myeloid subsets that are conserved between mouse and human lupus nephritis, thus providing a map and strong rationale for functional studies in mice with relevance to human disease.

immunology↗

bioRxiv2023

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy -- rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

biophysics↗

bioRxiv2023

ATP and Zn2+ induce folding of ALS-associated hSOD1 with fundamentally distinctive mechanisms

153-residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS, while wild-type hSOD1 aggregation is associated with SALS. So far >180 ALS-causing mutations have been identified within hSOD1, but the underlying mechanism still remains enigmatic. Mature hSOD1 is extremely stable constrained by a disulfide bridge to adopt a Greek-key {beta}-barrel fold that houses copper and zinc cofactors. Conversely, nascent hSOD1 is unfolded and prone to aggregation, requiring Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations disrupting Zn2+-binding correlate with their capacity to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provide not only mechanistic insights, but also therapeutic prospects for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations, encompassing 8 essential for humans (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, exhibited negligible binding or induction for folding of nascent hSOD1. Cu2+ displayed extensive binding to the unfolded state but induced severe aggregation. Unexpectedly, here for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing ability. Surprisingly, Zn2+ failed to induce folding of H80S/D83S-hSOD1, while Fe2+ could. By contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-induced folding of G93A-hSOD1. Comparing with ATP-induced folded state, our results delineate that hSOD1 maturation requires: 1) intrinsic folding capacity encoded by the sequence; 2) specific Zn2+-coordination; 3) disulfide formation and Cu-load catalyzed by hCCS. This study decodes a previously-unknown interplay of cations in controlling the initial folding of hSOD1, thus not only underscoring the critical role of Zn2+ in hSOD1-associated ALS, but also suggesting novel hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity likely relevant to other diseases and aging.

biophysics↗

bioRxiv2023

Functional archaic DNA regulates molecular variation and is associated with disease risk across global populations

The human genome contains many remnants of its evolutionary history, including a large number of evolutionarily volatile loci which have been introduced since our divergence from primates. One particularly intriguing source of novel DNA sequences is introgression events with archaic species which co-existed with modern humans. Both Neanderthals, who were common in Europe, and Denisovans, who have been observed only in Asia, have contributed genetic variants to the modern human genome but the functional consequences of these introgressed variants have yet to be investigated systematically. In this work, we show that Neanderthal and Denisovan DNA is most enriched for genetic variants which regulate gene expression in Europe and East Asia respectively, i.e. the populations in which the introgression event(s) most contributed to contemporary genetic variation. Neanderthal eQTLs, in particular, frequently upregulate gene expression. Archaic eQTLs from these two species regulate target genes with similar molecular functions which are distinct in each contemporary population, with the only common enrichment being for Neanderthal eQTLs to regulate taste receptor genes in both Europe and East Asia. We observed a correlated pattern of enrichment and depletion of medical phenotypes across Neanderthal and Denisovan eQTLs, including a shared enrichment for CNVs associated with developmental delay. Our results demonstrate the role of functional archaic DNA in regulating molecular phenotypes and disease risk across global populations and confirm the relevance of recently acquired DNA to contemporary human genetic variation. Author SummaryModern humans co-existed and interbred with two archaic human species (Neanderthals and Denisovans). The results of these events can still be detected as introgressed, archaic DNA sequences within the modern human genome. Here, we surveyed the contribution of functional archaic DNA across European and Asian populations by assessing their contribution to genetic variants which regulate gene expression in these two populations. We found that both species make a disproportionate functional contribution to the population with which they shared the most overlap (i.e. Neanderthals in Europe and Denisovans in East Asia). Although only Neanderthal DNA drives a higher level of gene expression compared to modern genetic variants, the DNA from both archaic species frequently regulates genes involved in many different biological processes and risk of disease, including a shared contribution to developmental delay. These results confirm the relevance of our recent evolutionary past in generating functional variation across global populations and the importance these recently introduced genetic sequences play in regulating current biological variation, such as disease risk.

genomics↗

bioRxiv2023

Embryonic type 3 innate lymphoid cells sense maternal dietary cholesterol to control local Peyer's patch development

Lymphoid tissue inducer (LTi) cells develop during intrauterine life and rely on developmental programs to initiate the organogenesis of secondary lymphoid organs (SLOs). This evolutionary conserved process endows the fetus with the ability to orchestrate the immune response after birth and to react to the triggers present in the environment. While it is established that LTi function can be shaped by maternal-derived cues and is critical to prepare the neonate with a functional scaffold to mount immune response, the cellular mechanisms that control anatomically distinct SLO organogenesis remain unclear. We discovered that LTi cells forming Peyers patches, gut-specific SLOs, require the coordinated action of two migratory G protein coupled receptors (GPCR) GPR183 and CCR6. These two GPCRs are uniformly expressed on LTi cells across SLOs, but their deficiency specifically impacts Peyers patch formation, even when restricted to fetal window. The unique CCR6 ligand is CCL20, while the ligand for GPR183 is the cholesterol metabolite 7,25-Dihydroxycholesterol (7,25-HC), whose production is controlled by the enzyme cholesterol 25-hydroxylase (CH25H). We identified a fetal stromal cell subset that expresses CH25H and attracts LTi cells in the nascent Peyers patch anlagen. GPR183 ligand concentration can be modulated by the cholesterol content in the maternal diet and impacts LTi cell maturation in vitro and in vivo, highlighting a link between maternal nutrients and intestinal SLO organogenesis. Our findings revealed that in the fetal intestine, cholesterol metabolite sensing by GPR183 in LTi cells for Peyers patch formation is dominant in the duodenum, the site of cholesterol absorption in the adult. This anatomic requirement suggests that embryonic, long-lived non-hematopoietic cells might exploit adult metabolic functions to ensure highly specialized SLO development in utero.

immunology↗

bioRxiv2023

Dopamine promotes Klebsiella quasivariicola proliferation and inflammatory response in the presence of macrophages

Klebsiella quasivariicola was a novel strain of Klebsiella species and had potential pathogenicity. Our previously studies showed dopamine, one of the most commonly used rescue drugs for critically ill patients, had clear effects on the growth of K. quasivariicola in culture medium, however, its effects on host immune system were ignored. Therefore, in consideration of the host immunity, the interactions of K. quasivariicola, dopamine and macrophages were explored. In this study, RAW264.7 cells and C57/BL6 mice were infected with K. quasivariicola, and the bacterial growth in macrophage, the production of inflammatory cytokines and the pathological changes of mice lung were detected, in the absence or presence of dopamine. Our results showed dopamine inhibited the K. quasivariicola growth in medium, but promoted the bacterial growth when K. quasivariicola was co-cultured with macrophages; The expression of proinflammatory cytokines in K. quasivariicola infected RAW 264.7 were increased, while a sharp rise was observed with the addition of dopamine; Infection of K. quasivariicola to mice induced an inflammatory response and lung injury, which were exacerbated by dopamine administration. It can be concluded that dopamine administration resulted in a significantly increase of K. quasivariicola burdens in the presence of macrophage, consequently, aggravated the inflammatory response and inflammatory injury. ImportanceDopamine is one of the most commonly used rescue drugs for critically ill patients. Here we indicated K. quasivariicola was a potential pathogen of pulmonary infection, and dopamine significantly increased the proliferation of K. quasivariicola when exposed to macrophage, subsequently result in severe inflammatory response and inflammatory injury. We also proposed an in vitro model of microbes-drugs-host immune cells that could better mimic in vivo environment and more suitable for the studies of inhibitor screening. This fundamental work had contributed to the present understanding of the crosstalk between pathogen, dopamine and host immune cells. Furthermore, our data showed dopamine was one of the risk factors for patients with K. quasivariicola infection, which provided a basis for clinical precision medicine.

microbiology↗

bioRxiv2023

Quantitative proteomics combined with network pharmacology analysis unveils the biological basis of Schisandrin B in treating diabetic nephropathy

BackgroundDiabetic nephropathy (DN) is a major complication of diabetes. Schisandrin B (Sch) is a natural pharmaceutical monomer that was shown to prevent kidney damage caused by diabetes and restore its function. However, there is still a lack of comprehensive and systematic understanding of the mechanism of Sch treatment in DN. ObjectiveWe aim to provide a systematic overview of the mechanisms of Sch in multiple pathways to treat DN in rats. MethodsStreptozocin was used to build a DN rat model, which was further treated with Sch. The possible mechanism of Sch protective effects against DN was predicted using network pharmacology and was verified by quantitative proteomics analysis. ResultsHigh dose Sch treatment significantly downregulated fasting blood glucose, creatinine, blood urea nitrogen, and urinary protein levels and reduced collagen deposition in the glomeruli and tubule-interstitium of DN rats. The activities of superoxide dismutase (SOD) and plasma glutathione peroxidase (GSH-Px) in the kidney of DN rats significantly increased with Sch treatment. In addition, the levels of IL-6, IL-1{beta}, and TNF- were significantly reduced in DN rats treated with Sch. 11 proteins that target both Sch and DN were enriched in pathways such as MAPK signaling, PI3K-Akt signaling, renal cell carcinoma, gap junction, endocrine resistance, and TNF signaling. Furthermore, quantitative proteomics showed that Xaf1 was downregulated in the model vs. control group and upregulated in the Sch-treated vs. model group. Five proteins, Crb3, Tspan4, Wdr45, Zfp512, and Tmigd1, were found to be upregulated in the model vs. control group and downregulated in the Sch vs. model group. Three intersected proteins between the network pharmacology prediction and proteomics results, Crb3, Xaf1, and Tspan4, were identified. ConclusionSch functions by relieving oxidative stress and the inflammatory response by regulating Crb3, Xaf1, and Tspan4 protein expression levels to treat DN disease.

pharmacology and toxicology↗

bioRxiv2022

Glucocerebrosidase activity and lipid levels are related to protein pathologies in Parkinson's disease

Parkinsons disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases characterized by the accumulation of misfolded -synuclein in the form of Lewy pathology. While most cases are sporadic, there are rare genetic mutations that cause disease and more common variants that increase incidence of disease. The most prominent genetic mutations for PD and DLB are in the GBA1 and LRRK2 genes. GBA1 mutations are associated with decreased glucocerebrosidase activity and lysosomal accumulation of its lipid substrates, glucosylceramide and glucosylsphingosine. Previous studies have shown a link between this enzyme and lipids even in sporadic PD. However, it is unclear how the protein pathologies of disease are related to enzyme activity and glycosphingolipid levels. To address this gap in knowledge, we examined quantitative protein pathology, glucocerebrosidase activity and lipid substrates in parallel from 4 regions of 91 brains with no neurological disease, idiopathic, GBA1-linked, or LRRK2-linked PD and DLB. We find that several biomarkers are altered with respect to mutation and disease severity. We found mild association of glucocerebrosidase activity with disease, but a strong association of glucosylsphingosine with -synuclein pathology, irrespective of genetic mutation. This association suggests that Lewy pathology precipitates changes in lipid levels related to disease severity.

neuroscience↗

bioRxiv2022

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 induction during cooling remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/514062v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@ef0a5org.highwire.dtl.DTLVardef@d907a5org.highwire.dtl.DTLVardef@85176aorg.highwire.dtl.DTLVardef@1c4306a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the {beta}2-adrenergic receptor ({beta}2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gs from {beta}2AR and G{beta}{gamma}, as opposed to a slow closing of the Gs -helical domain (AHD). Yeast-two-hybrid screening using Gs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gs AHD.

molecular biology↗