Biology subjects

Zhang, Q.

Publications and source records attributed to Zhang, Q..

At least 307 records · Page 17Linked to original sources

bioRxiv2024

Proteomic analysis reveals the dominant effect of ipomoeassin F on the synthesis of membrane and secretory proteins in triple-negative breast cancer cells

Ipomoeassin F (Ipom-F) is a natural compound with embedded carbohydrates that exhibits a potent cytotoxic effect on triple-negative breast cancer (TNBC) cells. The mechanism behind this selective potency remains unclear. To elucidate this mechanism, we analyzed the proteome profiles of the TNBC MDA-MB-231 cells after exposure to Ipom-F at different time points and increasing doses using a quantitative proteomic method. Our proteomic data demonstrate that the major effect of Ipom-F on MDA-MB-231 cells is the inhibition of membrane and secreted protein expression. Our proteomic data are consistent with the recently uncovered molecular mechanism of action of Ipom-F, which binds to Sec61- and inhibits the co-translational import of proteins into the endoplasmic reticulum. We have defined a subset of membrane and secreted proteins particularly sensitive to Ipom-F. Analysis of the expression of these Ipom-F-sensitive proteins in cancer cell lines and breast cancer tissues demonstrates that some of these proteins are upregulated in TNBC cells. Thus, it is likely that TNBC cells may have adapted to the elevated levels of some proteins identified as sensitive to Ipom-F in this study; inhibition of the expression of these proteins leads to a crisis in proliferation and/or survival for the cells.

cancer biology↗

bioRxiv2024

Structural Basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is the most widely investigated RNA-guided adaptive immune system in prokaryotes against the foreign genetic elements. Unlike the previously characterized Cas3 nuclease in the typical Type I-E system that exhibits uncontrolled progressive DNA cleavage, a recently discovered HNH domain linked to the Cas5 subunit functions as the nuclease for precise DNA cleavage, highlighting the potential of this new Type I-E variant system as a precise genome editing tool. Here, we present five near- atomic cryo-EM (cryo-electron microscopy) structures of Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex either bound or unbound to DNA. We revealed that the HNH domain extensively interacts with the adjacent subunits, including Cas6, Cas8 and Cas11. The related mutations of the crucial identified interactions can significantly weaken the performance of the enzyme. Upon the binding of DNA, the Cas-HNH/Cascade complex adopts a more compacted conformation with the subunits moving towards the center of the nuclease, thus activating the nuclease. We further identified four conserved cysteines that form a zinc-finger structure in the HNH domain and the mutations of these four cysteines can totally abolish the enzyme activity. Interestingly, we also discovered that the divalent ions such as zinc, cobalt and nickel down-regulate the enzyme performance by decreasing the stability of the Cascade complex. Together, our findings provide first structural insights into the assembly and activation of the Cas5-HNH/Cascade complex, opening new avenues for engineering this system for precise genome editing.

biophysics↗

bioRxiv2024

F-CPI: Prediction of activity changes induced by fluorine substitution using multimodal deep learning

There are a large number of fluorine (F)-containing compounds in approved drugs, and F substitution is a common method in drug discovery and development. However, F is difficult to form traditional hydrogen bonds and typical halogen bonds. As a result, accurate prediction of the activity after F substitution is still impossible using traditional drug design methods, whereas artificial intelligence driven activity prediction might offer a solution. Although more and more machine learning and deep learning models are being applied, there is currently no model specifically designed to study the effect of F on bioactivities. In this study, we developed a specialized deep learning model, F-CPI, to predict the effect of introducing F on drug activity, and tested its performance on a carefully constructed dataset. Comparison with traditional machine learning models and popular CPI task models demonstrated the superiority and necessity of F-CPI, achieving an accuracy of approximately 89% and a precision of approximately 67%. In the end, we utilized F-CPI for the structural optimization of hit compounds against SARS-CoV-2 3CLpro. Impressively, in one case, the introduction of only one F atom resulted in a more than 100-fold increase in activity (IC50: 22.99 nM vs. 28190 nM). Therefore, we believe that F-CPI is a helpful and effective tool in the context of drug discovery and design.

bioinformatics↗

bioRxiv2024

VPS28 regulates triglyceride synthesis and is mediated by the ubiquitination pathway in a bovine mammary epithelial cell line and mouse model

VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs), and is involved in ubiquitination. Ubiquitination is a crucial system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in regulating lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell (MAC-T) line and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results indicated that VPS28 knockdown significantly upregulated the fatty acid transporter CD36 (CD36 molecule) and the adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, alongside elevated expression of ubiquitin (UB) protein and reduced proteasome activity. In contrast, VPS28 overexpression increased CD36 levels without significantly affecting ADFP and TG levels, showing a trend toward reduced lipid droplets and increased UB expression and proteasome activity. Furthermore, the inhibition of the ubiquitin-proteasome system and endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, resulted in a further elevation of CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo by a mouse model, where VPS28 knockdown enhanced CD36, ADFP, UB expression, TG content, and lipid droplets in mammary glands, without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine in vitro cell model.

genetics↗

bioRxiv2024

Enhanced purinergic signaling in the paraventricular hypothalamus induces hyperphagic obesity and insulin resistance

Body energy homeostasis is tightly regulated by hypothalamic neural circuits. However, their remodeling upon metabolic challenges remains incompletely characterized, thus complicating the development of safe medications against the surge of metabolic diseases. Oxytocin (OXT) neurons in the paraventricular nucleus of the hypothalamus (PVH) are one of the key effectors regulating energy balance. In this work, we report that high-fat diet (HFD) feeding in mice evokes spatiotemporally selective ATP release from astrocytes (Inflares) in the PVH, accompanied by the expression of hematopoietic lineage-specific ADP/ATP receptor P2Y12 on local OXT (PVHOXT) neurons. Strikingly, increased purinergic signaling leads to impaired responsiveness of PVHOXT neurons accompanied by hyperphagic obesity and insulin resistance in mice. Conversely, loss of P2Y12 in PVHOXT neurons attenuates metabolic phenotypes, illustrating that such remodeling is both necessary and sufficient to induce metabolic phenotypes. Inflares were also induced by hyperglycemia, while emergence of P2Y12 on OXT neurons of patients with diabetes mellitus suggests an evolutionary conserved purinergic response to various metabolic challenges and its potential as a drug target. Accordingly, nasal administration of clinically approved doses of P2Y12 inhibitors counteracts obesity in mice and non-human primates, paving the way for application of these compounds in patients with metabolic disorders.

neuroscience↗

bioRxiv2024

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis

In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1-CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1s role in cell cycle control and oncogenesis beyond RB phosphorylation.

biochemistry↗

bioRxiv2024

Polymerization of Tspan7 into Helical Transmembrane Skeletons for Membrane Tether Stabilization

Tubular cell membrane protrusions, such as filopodia1, dendrites2, tunneling nanotubes (TNTs)3 and retraction fibers4, are prevalent on the cell surface. They play crucial roles in various physiological processes and disease states, including angiogenesis, wound healing, and cancer metastasis5-7. The shaping and stabilization of these protrusions are crucial for their function, and the molecular machineries involved have long been an active area of investigation. Here we employed an integrative approach combining live-cell imaging, in vitro reconstitution, and in situ cryo-EM analysis to reveal that the transmembrane protein tetraspanin 7 (TSPAN7) senses membrane curvature and polymerizes into a helical configuration on highly curved tubular membranes. These spirals act as a "transmembrane skeleton", effectively maintaining the structural integrity of membrane protrusions under mechanical stress. Our findings reveal a previously unreported assembly strategy for transmembrane proteins and a mechanism for maintaining tubular membrane protrusions.

cell biology↗

bioRxiv2024

Modular Fluorescent Cholesterol Naphthalimide Probes And Their Application For Cholesterol Trafficking Studies In Cells

Development of fluorescent cholesterol analogs to better understand subcellular cholesterol trafficking is of great interest for cell biology and medicine. Our approach utilizes a bifunctional 1,8-naphthalimide scaffold with a push-pull character, modified on one side with a head group and a linker on the other side connecting it to cholesterol via an ester bond. Through structure-function studies, weve explored how different substituents--linkers and head groups--affect the ability of these fluorescent cholesterol naphthalimide analogs (CNDs) to mimic natural cholesterol behavior at both molecular and cellular levels. We categorized the resulting analogs into three groups: neutral, charged, and those featuring a hydroxyl group. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Extensive all-atom molecular dynamics simulations helped us examine how these analogs perform in model membranes compared to cholesterol. Additionally, we investigated the partitioning of these fluorescent probes in phase-separated giant unilamellar vesicles. We evaluated the uptake and distribution of these probes within mouse fibroblast cells and astrocytes, for their subcellular distributions in lysosomes and compared that to BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of the fluorescent probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesterol probes bearing distinct spectral, biophysical, and cellular targeting features, which makes it a valuable toolkit for the investigation of subcellular distribution and trafficking of cholesterol and its derivatives.

biochemistry↗

bioRxiv2024

Mitochondrial defects and metabolic vulnerabilities in Lynch syndrome-associated MSH2-deficient endometrial cancer

Lynch syndrome (LS) is defined by inherited mutations in DNA mismatch repair genes, including MSH2, and carries 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, specific mechanisms for LS-associated endometrial carcinogenesis are not well understood. Here, we assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines established from this model, human tissues, and human EC cell lines with isogenic MSH2 knockdown. Beginning at eight months of age, 30% of Msh2KO mice exhibited endometrial atypical hyperplasia (AH), a precancerous lesion. At 12 to 16 months of age, 47% of Msh2KO mice exhibited either AH or ECs with histologic features similar to human LS-related ECs. Transcriptomic profiling of EC from Msh2KO mice revealed a transcriptomic signature for mitochondrial dysfunction. Studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: marked mitochondrial content reduction, along with pronounced disruptions to the integrity of retained mitochondria. Human LS-related ECs also exhibited mitochondrial content reduction compared with non-LS-related ECs. Functional studies revealed metabolic reprogramming of MSH2-deficient EC cells in vitro, including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. We are the first to identify mitochondrial dysfunction and metabolic disruption as a consequence of MSH2 deficiency-related EC. Mitochondrial and metabolic aberrations should be evaluated as novel biomarkers for endometrial carcinogenesis or risk stratification and could serve as targets for cancer interception in women with LS. SignificanceThis is the first study to report mitochondrial dysfunction contributing to MSH2-deficient endometrial cancer development, identifying a noncanonical pathway for MSH2 deficient carcinogenesis, which also imparts vulnerability to metabolic targeting.

cancer biology↗

bioRxiv2024

Observation of Higher-Order Assemblies Controlled by Protein One-dimensional Movement

RIG-I-like receptors (RLRs) detect cytosolic viral RNAs and initiate innate immune response. MDA5 is an RLR that recognizes viral long double-stranded RNA (dsRNA). The central helicase and C-terminal domains of MDA5 form a ring-like clamp around dsRNA, and its activation requires the oligomerization of N-terminal caspase activation and recruitment domains (CARDs). Another RLR, LGP2, lacks CARDs but plays essential roles in MDA5 signaling. However, the mechanisms underlying MDA5 CARDs assembly and the collaborative mechanics between MDA5 and LGP2 remain largely unresolved. Here we found the MDA5 ring-like clamp operates as an ATP-hydrolysis-driven motor, with the self-assembly of its CARDs strictly suppressed by the protein one-dimensional (1D) translocation along dsRNA. LGP2 recognizes a mobile MDA5 and inhibits the motors movement, thereby allowing MDA5 CARDs to assemble into oligomers that can further interact with downstream components. These findings highlight how RLR sensors orchestrate higher-order assemblies through 1D movement to direct immune response.

biophysics↗

bioRxiv2024

H2A.Z deposition at meiotic prophase I underlies homologous recombination and pachytene genome activation during male meiosis

Accurate meiotic progression is important for gamete formation and the generation of genetic diversity. However, little is known about the identity of chromatin regulators that underlie mammalian meiosis in vivo. Here, we identify the multifaceted functions of the chromatin remodeler ZNHIT1 in governing meiosis. The expression of Znhit1 gradually increases during the meiotic prophase, and Znhit1 knockout in spermatocytes results in arrested pachytene development, impaired DNA double-strand break repair, and defective homologous recombination. Single-cell RNA sequencing and transcriptome analysis reveal that Znhit1 loss dysregulates meiotic transcriptional programs at the pachytene stage. Chromatin immunoprecipitation data show that ZNHIT1 is needed for the incorporation of the histone variant H2A.Z into pachytene chromatin. Moreover, we found that H2A.Z cooperates with the transcription factor A-MYB to co-bind DNA elements and control gene activity. Our findings provide insights into the regulatory mechanisms governing meiotic progression and highlight ZNHIT1 as a critical regulator of meiotic progression.

developmental biology↗

bioRxiv2024

Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate ({beta}-HB). Prior work, based on detecting {beta}-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export {beta}-HB across the apical membrane. Here, we compare the accuracy of measuring {beta}-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of {beta}-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete {beta}-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming {beta}-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce {beta}-HB. Altogether, we substantiate {beta}-HB secretion in RPE but find that the secretion is equal apically and basally, RPE {beta}-HB can derive from ketogenic amino acids or fatty acids, and accurate {beta}-HB assessment requires mass spectrometric analysis.

cell biology↗

bioRxiv2024

Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used fresh frozen tissue samples from triple-negative breast cancer (TNBC) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse phase chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

cancer biology↗

bioRxiv2024

Dihydromyricetin promotes GLP-1 secretion to improve insulin resistance via "gut microbiota-CDCA"

Dihydromyricetin (DHM) is a polyphenolic phytochemical found mainly in plants such as Ampelopsis grossedentata, which has beneficial effects on insulin resistance. However, the specific mechanism has not been clarified. In this study, C57BL/6 mice were exposed to a high-fat diet (HFD) for eight weeks. DHM could improve insulin resistance via enhancing the incretin effect. DHM increased serum GLP-1 by improving intestinal GLP-1 secretion and inhibiting GLP-1 decomposition, associated with the alteration of intestinal intraepithelial lymphocytes (IELs) proportions and decreased expression of CD26 in IELs and TCR{beta}+ CD8{beta}+ IELs in HFD-induced mice. Meanwhile, DHM could ameliorate GLP-1 level and insulin resistance by modulation of gut microbiota and the metabolites, particularly the regulation of intestinal bile acid CDCA content, followed by the inhibition of FXR expression in intestinal L cells as well as increased Gcg mRNA expression and the secretion of GLP-1. These findings clarify the role of the "gut microbiota-CDCA" pathway in the improvement of intestinal GLP-1 levels in HFD-induced mice by DHM administration, providing a new pharmacological target for the prevention of insulin resistance.

pharmacology and toxicology↗

bioRxiv2024

Protective Effect of 2-Hydroxyestrone and2-Hydroxyestradiol Against Chemically-Induced Hepatotoxicity In Vitro and In Vivo

Ferroptosis is a form of regulated cell death closely associated with glutathione depletion and accumulation of reactive lipid peroxides. In the present study, we seek to determine whether 2-hydroxyestrone (2-OH-E1) and 2-hydroxyestradiol (2-OH-E2), two major metabolites of endogenous estrone and 17{beta}-estradiol formed in liver by cytochrome P450 enzymes, can strongly protect against erastin- and RSL3-induced ferroptosis in hepatoma cells (H-4-II-E and HuH-7) in vitro and acetaminophen-induced mouse liver injury in vivo. We find that 2-OH-E1 and 2-OH-E2 can protect, in a dose-dependent manner, H-4-II-E hepatoma cells against erastin/RSL3-induced ferroptosis. Similar protective effect of 2-OH-E1 and 2-OH-E2 against RSL3-induced ferroptosis is also observed in HuH-7 hepatoma cells. These two estrogen metabolites strongly reduce the levels of erastin- and/or RSL3-induced accumulation of cellular NO, ROS and lipid-ROS. Mechanistically, 2-OH-E1 and 2-OH-E2 protect cells against chemically-induced ferroptosis mainly by binding to cellular protein disulfide isomerase (PDI), and then inhibit its catalytic activity and reduce PDI-catalyzed formation of iNOS dimer, thereby abrogating cellular NO, ROS and lipid-ROS accumulation. Animal studies show that 2-OH-E1 and 2-OH-E2 can also exert a strong protection against acetaminophen-induced liver injury in mice. Interestingly, while estrone and 17{beta}-estradiol display a very weak protective effect in cultured hepatoma cells, they exert a similarly-strong protective effect as 2-OH-E1 and 2-OH-E2 in vivo, suggesting that the metabolic conversion of estrone and 17{beta}-estradiol to 2-OH-E1 and 2-OH-E2 contributes importantly to their hepatoprotective effect. The results of this study reveal that 2-OH-E1 and 2-OH-E2 are important endogenous factors for protection against chemically-induced liver injury in vivo.

pharmacology and toxicology↗

bioRxiv2024

The antimicrobial peptides pipeline: a bacteria-centric AMP predictor

Antimicrobial peptides (AMPs), unlike antibiotics, are encoded in genomes. AMPs are exported from the cell after expression and translation. In the case of bacteria, the exported peptides target other microbes to give the producing bacterium a competitive edge. While AMPs are sought after for their similar antimicrobial activity to traditional antibiotics, it is difficult to predict which combinations of amino acids will confer antimicrobial activity. Many computer algorithms have been designed to predict whether a sequence of amino acids will exhibit antimicrobial activity, but the vast majority of validated AMPs in databases are still of eukaryotic origin. This defies common sense since the vast majority of life on earth is prokaryotic. The antimicrobial peptides pipeline, presented here, is a bacteria-centric AMP predictor that predicts AMPs by taking design inspiration from the sequence properties of bacterial genomes with the intention to improve detection of naturally occurring bacterial AMPs. The pipeline integrates multiple concepts of comparative biology to search for candidate AMPs at the primary, secondary and tertiary peptide structure level. Results showed that the antimicrobial peptides pipeline identifies known AMPs that are missed by state-of-the-art AMP predictors, and that the pipeline yields more AMP candidates from real bacterial genomes than from fake genomes, with the rate of AMP detection being significantly higher in the genomes of seven nosocomial pathogens than in the fake genomes.

bioinformatics↗

bioRxiv2024

Use of machine learning for quantification of retinal pigment epithelium tight junctions improves assay sensitivity

The retinal pigment epithelium (RPE) is critical for maintaining outer retinal barrier homeostasis. In age-related macular degeneration (AMD), the RPE can undergo a dedifferentiation process that includes tight junction (TJ) loss and displacement of zonula occludens-1 (ZO-1), which may impair structural and functional integrity of the RPE barrier and contribute to disease pathogenesis. Our objective was to develop an automated and sensitive quantification method for TJ aberrations in an RPE immunofluorescence imaging assay, following treatment with TNF or TGF{beta}2. However, quantifying ZO-1 morphological changes in the RPE using standard image analysis methods did not provide a satisfactory assay window. To address this challenge, we developed an imaging assay to quantify ZO-1 changes using a machine learning approach, enabling enhanced phenotypic characterization of the ZO-1 changes in RPE cells and improved assay sensitivity. We were also able to capture and quantify the reversal of these changes using etanercept, an TNF inhibitor, with this imaging assay. Our findings indicated that this machine learning ZO-1 quantification assay could serve as a potential phenotypic readout for RPE dedifferentiation and enabling large-scale mechanistic studies.

bioinformatics↗

bioRxiv2024

Biomarker Identification by Proteomic Analysis of Vitreous Humor and Plasma in Diabetic Retinopathy

ImportanceIdentify detectable plasma and/or vitreous signals to potentially predict diabetic retinopathy (DR) progression for earlier disease intervention. ObjectiveTo determine the mediators and potential disease progression biomarkers of DR in vitreous humor (VH) and plasma samples using the SomaScan proteome profiling platform. DesignDifferential expression analysis was conducted on VH and plasma samples using the SomaScan Assay. SettingA non-interventional study conducted to collect and analyze VH and plasma samples from patients with diabetic retinopathy. ParticipantsSamples from DR (60 nonproliferative diabetic retinopathy/NPDR, 60 proliferative diabetic retinopathy/PDR) and 60 control patients were collected. Main outcomes and MeasuresDifferentially expressed proteins between disease and control groups were identified. Pathway enrichment analysis was conducted to identify significantly perturbed pathways in DR. Finally, a random forest model was used to identify predictive biomarkers of disease progression. ResultsSomaScan v3 is a pooled aptamer hybridization assay using 5080 SOMAmers to probe over 4100 proteoforms in VH and plasma samples from 3 groups (control, NPDR, and PDR). The most profound protein content change was observed in the VH samples of PDR patients, while minimal changes were measured in plasma samples, highlighting the regionality of PDR pathogenesis. Many key molecules and molecular pathways such as VEGF-A, erythropoietin, and inflammation-associated proteins implicated in DR were significantly affected in the VH of PDR patients. In addition to the classic pathways (hypoxia, immune response, mTORC1 signaling) known to be involved in PDR, novel signaling pathways, including HEME metabolism and adipogenesis, were identified in VH samples. Application of a machine learning algorithm identified a panel of plasma PDR predictive biomarkers and revealed SCARA5 as the top one based on the largest average Gini decrease in the model. ConclusionOur study identified profound alteration of protein expression and molecular pathways in the VH of PDR patients, supporting the key role of local pathogenic changes in DR progression compared to systemic factors. Although the systemic changes related to DR were small, a few disease progression predictive candidate biomarkers (SCARA5, PTK7, FAM3Band FAM3D) were identified, prompting further investigation. Key PointsQuestion: Are plasma/ vitreous humor (VH) proteins predictive of diabetic retinopathy (DR) progression? Findings: This study identifies substantial protein changes in the VH of proliferative diabetic retinopathy (PDR) patients, while early nonproliferative DR (NPDR) patients show minimal change. We identify multiple proteins linked to angiogenesis, inflammation, immune cells (microglia/macrophage/neutrophil), and leukostasis associated with PDR and reveal a potential plasma panel of disease progression (from NPDR to PDR) biomarkers (SCARA5, PTK7, FAM3B, FAM3D). Meaning: Identified disease progression predictive biomarkers permits potential development of prognostic tools to identify individuals most at risk for PDR progression and offering reduced disease burden by earlier intervention.

physiology↗