Human Sample Collection
The participants were hospitalized patients with PDR and cataract between November 2020 and November 2022 at the Tianjin Medical University Eye Hospital in Tianjin, China. PDR was based on the American Academy of Ophthalmology criteria (2019). Exclusion criteria were severe diabetic complications, severe and chronic infection symptoms, type 1 diabetes mellitus, acute liver and kidney dysfunction, malignant tumors, acute cardiovascular diseases, and patients with eye diseases other than cataracts. Eventually, 30 patients with PDR and 29 with cataracts were included. The anthropometric and biochemical malignant tumors of the participants are presented in Supplementary Table S1. Patients with cataracts underwent fundus photography, which confirmed no abnormalities in the fundus. Informed consent was obtained from all participants prior to the survey, and the Research Ethics Committee of Tianjin Medical University Eye Hospital approved the study.
Animals
Eight-week-old male C57BL/6J mice (8 weeks old) were purchased from SiPiFu (Beijing, China). All mice were housed within ventilated cages in a specific pathogen-free room at a constant temperature (23°±2°C) under a 12-hour light-dark cycle at the Tianjin Medical University Eye Institute Laboratory Animal Center (Tianjin, China). The animals were categorized into four groups: (1) control for T1DM, (2) T1DM (streptozotocin [STZ]-induced), (3) control for T2DM, and (4) T2DM (a high-fat diet [HFD] along with a low dose of STZ). Mice were starved for 12 h before the experiment. The control mice of the T1DM group were induced by intraperitoneal injection of 0.1 M sodium citrate buffer (Solarbio, Beijing, China) for 5 days. In contrast, the T1DM mice were induced with STZ (40 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) for 5 days. T2DM group’s control mice were given a normal diet (10 kcal% fat, D12450J, Research Diets, Keaoxieli, Beijing, China) for 14 days before being starved and injected with 0.1 M sodium citrate buffer. T2DM mice were fed a high-fat diet (HFD) (60 kcal% fat, D12492, Research Diets, SaiNo, Jilin, China) for 14 days and were then induced with a single dose of STZ (85 mg/kg), followed by maintaining on an HFD. Subsequent blood glucose levels were measured by expressing a small amount of blood from the lateral tail vein after puncture with a 22-gauge needle and analyzing the sample using a blood glucose meter (ACCU-CHEK; Roche, Basel, Switzerland). Mice with plasma glucose levels > 16.7 mmol/L were considered diabetic.
Adenovirus (Ad) vectors containing a null transgene (Ad-null) or a KL transgene (Ad-KL) were purchased from Scilia (Beijing, China). The diabetic group was further categorized into four subgroups: T1DM + Ad-null, T1DM + Ad-KL, T2DM + Ad-null, and T2DM + Ad-KL. Diabetic mice receiving KL treatment were injected with 2.5 × 109 plaque-forming units of Ad-KL via the tail vein, and Ad-null was injected as a control. The adenoviral transfer was repeated monthly after the first dose. The mice were euthanized 12 weeks after the first adenovirus injection (Fig S2).
Vldlr−/− mice were provided by Jackson Laboratory. The mouse pups were treated with an intravitreal injection of soluble KL protein (3 pmol/L) or PBS from P8 to P15. At P16, neovascular lesions were quantified.
Cell Culture
RAW264.7 lineage cells were cultivated with DMEM medium (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin and 100 mg/mL streptomycin (Beyotime, Shanghai, China) under 37°C in a 5% carbon dioxide environment in a humid incubating device. The cells were cultured in 6-well plates and transfected with Ad-KL. Cells (1 × 106 cells/well) were infected with the virus at an MOI of 100. After the cells were treated with the virus for 24 h, 4-hydroxynonenal (4HNE) (Cayman Chemical, Ann Arbor, MI), 10 µmol/L was administrated for 72 h, and the cells were harvested.
Wound Healing
RAW264.7 cells (1×106 cells / well) were seeded in a 6-well culture plate for 24 h, until they reached a confluence of 90%. Scratched wound lines were created using a 10 µL micropipette tip. Cells were treated for 24 h in serum-free medium with or without 50 ng/mL soluble KL protein (Cloud-Clone Corp.) and cultured for 24 h. The Scratched wound lines were examined using an Olympus microscope (DP73; Olympus, Tokyo, Japan), and calculated using the ImageJ software.
SA-β-Gal Staining
Staining for SA-β-gal activity was performed according to the manufacturer’s instructions (C0602; Beyotime, Shanghai, China). Briefly, cells and retinal sections were fixed in a fixative solution and stained in a staining working solution containing X-gal for 24 h at 37°C. Imaging was observed using an Olympus microscope, where senescent cells were stained blue.
Isolation of Mouse Peripheral Blood Mononuclear Cells (PBMCs)
Blood samples were collected by enucleation of eyeballs, and then PBMCs were isolated by a mouse peripheral blood monocyte isolation kit (Tbdscience, Tianjin, China). Briefly, the mononuclear cell layer was collected after density gradient centrifugation, washed three times with wash buffer, and centrifuged at 250 g for 10 min at room temperature to obtain mononuclear cell precipitates. The resulting mononuclear cell precipitate was used for protein extraction.
3D Structure Preparation and Molecular Docking of HECTD1 and IRS1
Molecular docking of HECTD1 and IRS1 crystal structures was performed to investigate protein-protein interactions. The solved crystal structures of the HECTD1 solution NMR structure (PDB ID 2LC3) and the IRS1 PTB domain (PDB ID 1IRS) were obtained from the UniProt database (https://www.uniprot.org/). Protein-protein molecular docking of HECTD1 and IRS1 was performed using ClusPro 2.0. Finally, images of the interactions were prepared using PyMOL 2.5.
Bioinformatics Analyses
The dataset GSE26168 was downloaded from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). Samples from nine T2DM samples and eight healthy controls were analyzed. GEO2R was used to explore differentially expressed genes (DEGs) between T2DM and normal blood samples, and heat maps were generated using ggplot2. Statistically significant DEGs were defined as |logFC| ≥1, and a P-value < 0.05 was the cut-off criterion. Senescence-related genes obtained from the Aging Atlas were 500 (https://ngdc.cncb.ac.cn/aging/age_related_genes). The protein-protein interaction (PPI) network of differentially expressed senescence-related genes was examined using the Search Tool for the Retrieval of Interacting Genes (STRING) database (https://string-db.org/). The PPI network was visualized and constructed using Cytoscape v3.8.2 software. Finally, 10 hub genes were screened by the cytoHubba plugin.
Data Independent Acquisition (DIA) Quantitative Proteomics
Six samples consisting of three biological replicates of two groups, Ad-null-transfected RAW264.7 cells, were prepared. Proteins were extracted using lysis buffer containing 8 mol/L urea, 50 mmol/L NH4HCO3, and 0.2% SDS, followed by 5 min of ultrasonication on ice. The lysate was centrifuged at 14,000× g for 10 min at 4°C, and the supernatant was moved to a clean tube. The protein concentration was measured using the bicinchoninic acid method, and 100 ug of protein was extracted from each sample. The samples were further reduced with 10 mM DTT for 1 h, then alkylated with 40 mM IAA for 1 h at room temperature in the dark. The protein was digested with trypsin at a ratio of 1:40 for 12–16 h at 37°C. Samples were centrifuged at 14000 rcf for 20 min at 4°C, and digested peptides were collected using 50 µL NH4HCO3 in three subsequent elution steps. Formic acid (1%, 7.5 µL) was added to the mixed digested peptides to stop digestion, and samples were dried under vacuum at 60°C for 45 min. The peptides were resuspended in 12 µL of 0.1% formic acid. Peptide concentration was measured using a Nanodrop spectrophotometer (Thermo Scientific, Basel, Switzerland) at an absorbance of 280 nm; 4 µg peptides were separated on a 30 min LC gradient using a Trap column (AB SCIEX, 10 × 0.3 mm, 5 µm 120 A C18 particles) and injected into Triple TOF 6600 (SCIEX, Framingham, MA, USA) mass spectrometer. The DIA acquisition scheme consisted of 96 variable windows ranging from 100 to 1500 m/z. Raw data from DIA quantitative proteomics were analyzed using Spectronaut 15 and searched against the UniProt Macaca mulatta database (https://www.uniprot.org/proteomes/UP000006718). A confidently identified protein requires at least one unique peptide with an FDR < 1%. We defined proteins with |logFC| ≥1.5 and a P-value < 0.05 as differentially expressed proteins (DEPs). Heatmaps were generated using the “pheatmap” package in R, and volcano plots using “ggplot2.” Gene ontology (GO) pathways were searched using the “clusterProfiler” package in R.
Query for E3-HECTD1 Interactions in UbiBrowser 2.0
UbiBrowser 2.0 (http://ubibrowser.bio-it.cn/ubibrowser_v3/) is a comprehensive database that predicts proteome-wide E3-substrate networks based on a naïve Bayesian classifier and combines various types of heterogeneous biological evidence. It currently contains 4068 known E3-substrate interactions in 38 organisms. This was used to predict the potential substrates of HECTD1.
Measurement of KL Level by Enzyme-Linked Immunosorbent Assay (ELISA)
Human KL ELISA kit was obtained from ImmunoBiological Laboratories (Takasaki, Japan). The ELISA was performed according to the manufacturer’s instructions. Briefly, 100 µL of EIA buffer, prepared standard and serum and aqueous humor samples were added to appropriate wells. The resulting plate was incubated for 60 min at room temperature with plate lid and subsequently washed four times with wash buffer. After removing all liquid completely, 100 µL of labeled antibody was added to each well. The resulting plate was incubated for 30 min at room temperature with a plate lid and subsequently washed four times with wash buffer. After washing the wells, 100 µL of TMB solution was added and incubated for 30 min at room temperature. Finally, the reaction was stopped by adding 100 µL stop solution, and the signal was measured at 450 nm.
RNA Interference Analysis
A small interfering RNA of HECTD1 (HECTD1-siRNA) was purchased from GenePharma (Shanghai, China). RAW264.7 cells were seeded in a 6-well plate with DMEM for 12 h before the transfection. The cells were then transfected with HECTD1-siRNA and negative control siRNA (NC-siRNA) for 72 h using Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA) in Opti-MEM medium (Thermo Fisher Scientific, MA, USA). The sequences are as follows:
HECTD1 siRNA: F: 5′-GCCAUCUACUUCAAGUCAATT-3′; R: 5′-UUGACUUGAAGUAGAUGGCTT − 3′.
Negative control: F: 5′-UUCUCCGAACGUGUCACGUTT-3′; R: 5′- ACGUGACACGUUCGGAGAATT − 3′.
Western Blot Analysis
Cells were sonicated using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China), and then centrifuged at 14,000 rpm for 30 min at 4°C. Protein concentrations in the supernatants were detected using the bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China). Next, 20 µg of protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride; membranes were blocked with a blocking buffer (Epizyme, Shanghai, China) for 20 min, and then incubated with the primary antibodies overnight at 4°C. Membranes were washed and incubated with either HRP-conjugated goat anti-rabbit or anti-mouse IgG for 2 h at room temperature. The antibodies used in this study are listed in Supplementary Table S2. Signals were developed using enhanced chemiluminescence substrate (Amersham, Buckinghamshire, U.K.) and visualized using autoradiography (Tanon, Shanghai, China). The gray density of the bands was performed using ImageJ software (NIH, Bethesda, MD, USA).
Immunoprecipitation and Ubiquitylation Assays
HECTD1 knockout RAW264.7 cells were lyzed and combined with 6 µg of anti-IRS1 (proteintech, Wuhan, China) per sample at 4°C overnight. Next, the antigen sample/antibody mixture was added to the tube containing 25 µL Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h with mixing. The beads were incubated with 100µl 1X lane marker sample buffer at 100ºC for 10 minutes and then Western blotting with anti-ubiquitin (PTM BIO, Hangzhou, China) was performed.
Immunofluorescence Assay
The cell slides and retinal flat mounts were fixed with 4% paraformaldehyde for 20 min. Cell and tissue slides were blocked with 0.2% bovine serum albumin (BSA) (Beyotime, Shanghai, China) and 5% goat serum (Gibco; Thermo Fisher Scientific, Massachusetts) in 0.3% Triton X-100 (Solarbio, Beijing, China) for 30 min at 4°C. The diluent primary antibody was incubated overnight at 4°C. After a washing step, the slides were incubated with diluent secondary antibodies conjugated to Alexa Fluor 488/594 for 2 h at room temperature. After counterstaining with DAPI, the slides were mounted in an antifade mounting medium (Solarbio, Beijing, China) and observed under a confocal microscope (Zeiss, Germany). The primary antibodies used are listed in Supplementary Table S2.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total mRNA was isolated from RAW264.7 using an RNA extraction kit (EZBioscience, US). The concentration of total RNA was measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA, MA) and was reverse-transcribed into cDNA using the color reverse transcription reagents (EZBioscience, US). Gene-specific primers were designed using the Primer-BLAST (Supplementary Table S3). Each qPCR reaction was performed using 2uL of cDNA and 1.5 µL of each primer in 5 µL of 2X Color SYBR Green Master Mix (EZ Bioscience, US). The cycling conditions were as follows: 5 min at 95°C, 40 cycles for 10 s at 95°C, and 30 s at 60°C. The SYBR Green fluorescence signal was measured at the end of each cycle using a LightCycler 480 II (Roche, Basel, Switzerland).
Electroretinography (ERG)
Mice were anesthetized with 5% chloral hydrate after at least 12 h of dark adjustment. Their pupils were fully dilated with tropicamide, and topical anesthesia was administered before recording began. Electrical responses were recorded using a forehead reference electrode and a ground electrode in the tail. Ganzfeld stimuli were delivered using Phoenix Micron IV (Phoenix Technologies, Beaverton, USA). Dark-adapted ERGs (1.0 cd. s/m² flash stimuli) were recorded.
Optical Coherence Tomography (OCT)
The thickness of each layer of mouse retina was measured using a Spectralis OCT (Heidelberg, Germany). Animals were anesthetized with 5% chloral hydrate. During sedation, tropicamide eye drops were administered to dilate the pupil. OCT images were acquired using 768 A scans, 30 B scans, and a 30-degree OCT field. Total retinal thickness and thickness of separate retinal layers were analyzed using the device’s internal software (Heidelberg Eye Explorer Software).
Statistical Analysis
All experiments were repeated at least thrice. All statistical analyses were performed with the Prism software (GraphPad, San Diego, CA, USA). Data are presented as standard error of the mean (SEM). All groups were normalized to the control group unless otherwise stated, and statistical analysis was carried out with unpaired Student’s t-tests, analysis of variance (ANOVA), and chi-square tests. Results were considered significant when P < 0.05.