Protein Transport Research Articles

Endosomal sorting protein SNX4 limits synaptic vesicle docking and release.

Sorting nexin 4 (SNX4) is an evolutionary conserved organizer of membrane recycling. In neurons, SNX4 accumulates in synapses, but how SNX4 affects synapse function remains unknown. We generated a conditional SNX4 knock-out mouse model and report that SNX4 cKO synapses show enhanced neurotransmission during train stimulation, while the first evoked EPSC was normal. SNX4 depletion did not affect vesicle recycling, basic autophagic flux, or the levels and localization of SNARE-protein VAMP2/synaptobrevin-2. However, SNX4 depletion affected synapse ultrastructure: an increase in docked synaptic vesicles at the active zone, while the overall vesicle number was normal, and a decreased active zone length. These effects together lead to a substantially increased density of docked vesicles per release site. In conclusion, SNX4 is a negative regulator of synaptic vesicle docking and release. These findings suggest a role for SNX4 in synaptic vesicle recruitment at the active zone.

Microglial CD2AP deficiency exerts protection in an Alzheimer’s disease model of amyloidosis

BackgroundThe CD2-associated protein (CD2AP) was initially identified in peripheral immune cells and regulates cytoskeleton and protein trafficking. Single nucleotide polymorphisms (SNPs) in the CD2AP gene have been associated with Alzheimer’s disease (AD). However, the functional role of CD2AP, especially its role in microglia during AD onset, remains elusive.MethodsCD2AP protein levels in cultured primary cells and in 5xFAD mice was studied. Microglial CD2AP-deficient mice were crossed with 5xFAD mice and the offspring were subjected to neuropathological assessment, behavioral tests, electrophysiology, RNA-seq, Golgi staining, and biochemistry analysis. Primary microglia were also isolated for assessing their uptake and morphology changes.ResultsWe find that CD2AP is abundantly expressed in microglia and its levels are elevated in the brain of AD patients and the 5xFAD model mice at pathological stages. We demonstrate that CD2AP haploinsufficiency in microglia significantly attenuates cognitive and synaptic deficits, weakens the response of microglia to Aβ and the formation of disease-associated microglia (DAM), and alleviates synapse loss in 5xFAD mice. We show that CD2AP-deficient microglia exhibit compromised uptake ability. In addition, we find that CD2AP expression is positively correlated with the expression of the complement C1q that is important for synapse phagocytosis and the formation of DAM in response to Aβ deposition. Moreover, we reveal that CD2AP interacts with colony stimulating factor 1 receptor (CSF1R) and regulates CSF1R cell surface levels, which may further affect C1q expression.ConclusionsOur results demonstrate that CD2AP regulates microgliosis and identify a protective function of microglial CD2AP deficiency against Aβ deposition, suggesting the importance of detailed investigation of AD-associated genes in different brain cells for thoroughly understanding their exact contribution to AD.

Integration analysis of microRNAs as potential biomarkers in early-stage lung adenocarcinoma: the diagnostic and therapeutic significance of miR-183-3p

IntroductionLung adenocarcinoma (LUAD) poses a significant therapeutic challenge, primarily due to delayed diagnosis and the limited efficacy of existing treatments.MethodsTo understand the pathogenesis and identify diagnostic biomarkers for LUAD in the early stage, we investigated differential miRNA expression in 33 stage I LUAD patients between tumor and matched paracancerous tissues by Illumina Sequencing. Target genes of differentially expressed miRNAs were predicted using TargetScan and miRDB databases and further analyzed by GO and KEGG pathway enrichment analysis. The miRNAs expression results were verified using qRT-PCR. Additionally, we evaluated the clinical significance of miRNAs by the TCGA database. miR-183-3p was chosen for subsequent biological functional studies by cell proliferation assays, cell migration and cell invasion assays, cell apoptosis and cell cycle assays in LUAD cells. The clinical relevance target genes of miR-183-3p were predicted by TargetScan databases and bioinformatics assays. Gene-specific experimental validation was performed using qRT-PCR, western blotting and luciferase reporter assays.ResultsWe identified 36 differentially expressed miRNAs between LUAD tissues and matched paracancerous tissues. Target genes for these miRNAs revealed associations with processes and pathways such as RNA biosynthesis, intracellular signaling, protein transport, and the Ras, MAPK, and PI3K-AKT pathways. The qRT-PCR results were in alignment with the sequencing data for 19 out of these 21 miRNAs which not yet implicated in LUAD, 13 were up-regulated, 6 were down-regulated. The clinical relevance assays showed that 5 up-regulated miRNAs have diagnostic value for LUAD. miR-183-3p showed significant advantages in the result of sequencing, qRT-PCR, and clinical relevance assay. Biological functional assays showed that miR-183-3p emerged as a key regulator, promoting LUAD cell proliferation, decreasing apoptosis, and augmenting migration and invasion capabilities. The clinical relevance assays and experimental validation showed SESN1 as a clinical significance target of miR-183-3p.DiscussionOur study lays the foundation for investigating miRNAs with diagnostic significance in early-stage LUAD, pointing out that inhibition of miR-183-3p may serve as a novel therapeutic in LUAD.

Drug Interaction Studies of Cabamiquine:Ganaplacide Combination against Hepatic Plasmodium berghei.

New antimalarial combination therapies with novel modes of action are required to counter the emergence and spread of Plasmodium drug resistance against existing therapeutics. Here, we present a study to evaluate the preventive activity of a combination of clinical antimalarial drug candidates, cabamiquine and ganaplacide, that have multistage activity against the liver and blood stages of Plasmodium infection. Cabamiquine (DDD107498, M5717) inhibits parasite protein synthesis, and ganaplacide (KAF156) inhibits protein trafficking, blocks the establishment of new permeation pathways, and causes endoplasmic reticulum expansion. The pharmacodynamic parameters of a combination of the two compounds were assessed employing a pharmacometrics approach in conjunction with in vitro-in silico checkerboard analysis. The in vitro study was performed on a previously established 3D infection platform based on human hepatic cell lines that sustain infection by rodent P. berghei parasites. The in vivo efficacy of this drug combination was assessed against the liver stage of the P. berghei. Our results show that the combination of both drugs at the tested concentrations does not interfere with the drugs respective mode of action or affect hepatocyte cell viability. The drug combination was fully effective in preventing the appearance of blood stage parasites when a systemic plasma Cav0-24/EC50 ratio >2 for ganaplacide and >5 for cabamiquine was achieved. These findings demonstrate that chemoprevention using a combination of cabamiquine and ganaplacide has the potential to target the asymptomatic liver stage of Plasmodium infection and prevent the development of parasitemia.

Update on NKCC2 regulation in the thick ascending limb (TAL) by membrane trafficking, phosphorylation, and protein-protein interactions

Purpose of reviewThe thick ascending limb (TAL) of loop of Henle is essential for NaCl, calcium and magnesium homeostasis, pH balance and for urine concentration. NKCC2 is the main transporter for NaCl reabsorption in the TAL and its regulation is very complex. There have been recent advancements toward understanding how NKCC2 is regulated by protein trafficking, protein-protein interaction, and phosphorylation/dephosphorylation. Here, we update the latest molecular mechanisms and players that control NKCC2 function, which gives an increasingly complex picture of NKKC2 regulation in the apical membrane of the TAL.Recent FindingsProtein-protein interactions are required as a regulatory mechanism in many cellular processes. A handful of proteins have been recently identified as an interacting partner of NKCC2, which play major roles in regulating NKCC2 trafficking and activity. New players in NKCC2 internalization and trafficking have been identified. NKCC2 activity is also regulated by kinases and phosphatases, and there have been developments in that area as well.SummaryHere we review the current understanding of apical trafficking of NKCC2 in the thick ascending limb (TAL) which is tightly controlled by protein-protein interactions, protein turnover and by phosphorylation and dephosphorylation. We discuss new proteins and processes that regulate NKCC2 that have physiological and pathological significance.

MamF-like proteins are distant Tic20 homologs involved in organelle assembly in bacteria

Organelle-specific protein translocation systems are essential for organelle biogenesis and maintenance in eukaryotes but thought to be absent from prokaryotic organelles. Here, we demonstrate that MamF-like proteins are crucial for the formation and functionality of bacterial magnetosome organelles. Deletion of mamF-like genes in the Alphaproteobacterium Magnetospirillum gryphiswaldense results in severe defects in organelle positioning, biomineralization, and magnetic navigation. These phenotypic defects result from the disrupted targeting of a subset of magnetosomal proteins that contain C-terminal glycine-rich integral membrane domains. Phylogenetic analyses reveal an ancient evolutionary link between MamF-like proteins and plastidial Tic20. Our findings redefine the molecular roles of MamF-like proteins and suggest that organelle-specific protein targeting systems also play a role in bacterial organelle formation.

H5 subtype avian influenza virus induces Golgi apparatus stress response via TFE3 pathway to promote virus replication.

During infection, avian influenza virus (AIV) triggers endoplasmic reticulum (ER) stress, a well-established phenomenon in previous research. The Golgi apparatus, situated downstream of the ER and crucial for protein trafficking, may be impacted by AIV infection. However, it remains unclear whether this induces Golgi apparatus stress (GAS) and its implications for AIV replication. We investigated the morphological changes in the Golgi apparatus and identified GAS response pathways following infection with the H5 subtype AIV strain A/Mallard/Huadong/S/2005. The results showed that AIV infection induced significant swelling and fragmentation of the Golgi apparatus in A549 cells, indicating the presence of GAS. Among the analyzed GAS response pathways, TFE3 was significantly activated during AIV infection, while HSP47 was activated early in the infection process, and CREB3-ARF4 remained inactive. The blockade of the TFE3 pathway effectively inhibited AIV replication in A549 cells and attenuated AIV virulence in mice. Additionally, activation of the TFE3 pathway promoted endosome acidification and upregulated transcription levels of glycosylation enzymes, facilitating AIV replication. These findings highlight the crucial role of the TFE3 pathway in mediating GAS response during AIV infection, shedding light on its significance in viral replication.

A New Severe Congenital Neutropenia Syndrome Associated with Autosomal Recessive COPZ1 Mutations.

We have identified a new inherited bone marrow (BM) failure syndrome with severe congenital neutropenia (CN) caused by autosomal recessive mutations in the coatomer protein complex I (COPI) subunit zeta 1 (COPZ1) gene. A stop-codon COPZ1 mutation and a missense mutation were found in three patients from two unrelated families. While two affected siblings with a stop-codon COPZ1 mutation suffered from congenital neutropenia (CN) that involves other hematological lineages, and non-hematological tissues, the patient with a missense COPZ1 mutation had isolated neutropenia. Both COPZ1 mutations were localized to a highly evolutionarily conserved region. The resulting truncated COPZ1 protein was predicted to display diminished interaction with its COPI complex partner, COPG1. These findings were consistent with the observed block in retrograde protein transport from the Golgi to the ER in human fibroblasts carrying truncated COPZ1. Human CD34+ cells with truncated or missense COPZ1 had significantly impaired granulocytic differentiation and in zebrafish embryos, truncated Copz1 also resulted in defective myelopoiesis. Intracellularly, truncated COPZ1 downregulated JAK/STAT/CEBPE/G-CSFR signaling and hypoxia-responsive pathways while inducing STING, interferon-stimulated genes, stimulating oxidative phosphorylation activity, and increasing reactive oxygen species (ROS) levels in CD34+ cells. Missense COPZ1 deregulated interferon and JAK/STAT signaling but less than the truncated protein. Finally, treatment with the small molecule HIF1α activator IOX2 or transduction of cells with COPZ2 cDNA restored defective granulopoiesis in COPZ1-mutated CD34+ cells, offering potential therapeutic options.

Discovery and validation of Hsa-microRNA-3665 promoter methylation as a potential biomarker for the prognosis of esophageal squaous cell carcinoma.

Methylation of microRNA (miRNA) promoters associated with diseases is a common epigenetic mechanism in the development of various human cancers. However, its relationship with prognosis in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to explore the association between the methylation level of has-miR-3665 promoter and prognosis in ESCC. Human miRNA data were downloaded from miRbase, and we identified CpG islands of these human miRNAs by genomics browser analysis. MiRNA methylation levels were detected by methylation-specific high-resolution melting. Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to explore the molecular mechanism of hsa-miR-3665. Cox regression analysis was used to investigate prognostic factors. The overall survival rate was predicted by a nomogram. We found that 88 human miRNAs had promoter methylatio, of which 15 miRNAs were found to be epigenetically regulated in ESCC cells compared with their normal counterparts, including hsa-miR-3665. Meanwhile, hsa-miR-3665 expression was significantly lower in ESCC tumour tissue than in adjacent tissue (P = 0.03). GO and KEGG analyses demonstrated that the target genes are involved in protein transport, transcription regulator activity, MAPK and RAS signaling pathway. High hsa-miR-3665 promoter methylation levels were associated with a poor prognosis (HR = 3.89, 95% CI 1.11 ~ 13.55). Moreover, a nomogram incorporating the hsa-miR-3665 methylation level and clinical factors presented a good performance for predicting survival in the training and validation tests, with C-indices of 0.748 and 0.751, respectively. High hsa-miR-3665 promoter methylation levels may be a potential biomarker for the progression of ESCC.

BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways.

We identify BEACH domain-containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.

Materials approaches for next-generation encapsulated cell therapies

AbstractTransplanted cells can act as living drug factories capable of secreting therapeutic proteins in vivo, with applications in the treatment of Type 1 diabetes (T1D), blood borne disease, vision disorders, and degenerative neural disease, potentially representing functional cures for chronic conditions. However, attack from the host immune system represents a major challenge, requiring chronic immunosuppression to enable long-lived cell transplantation in vivo. Encapsulating cells in engineered biomaterials capable of excluding components of the host immune system while allowing for the transport of therapeutic proteins, oxygen, nutrients, metabolites, and waste products represents a potential solution. However, the foreign-body response can lead to isolation from native vasculature and hypoxia leading to cell death. In this prospective article, we highlight materials-based solutions to three important challenges in the field: (i) improving biocompatibility and reducing fibrosis; (ii) enhancing transport of secreted protein drugs and key nutrients and oxygen via engineered, semipermeable membranes; and (iii) improving oxygenation. These efforts draw on several disciplines in materials’ research, including polymer science, surfaces, membranes, biomaterials’ microfabrication, and flexible electronics. If successful, these efforts could lead to new therapies for chronic disease and are a rich space for both fundamental materials’ discovery and applied translational science. Graphical Abstract

Polymeric nanoparticles for protein and peptide delivery

Protein- and peptide-based medications are recognized for their effectiveness and lower toxicity compared to chemical-based drugs, making them promising therapeutic agents. However, their application has been limited by numerous delivery challenges. Polymeric nanostructures have emerged as effective tools for protein delivery due to their versatility and customizability. Polymers’ inherent adaptability makes them ideal for meeting the specific demands of protein-delivery systems. Various strategies have been employed, such as enzyme inhibitors, absorption enhancers, mucoadhesive polymers, and chemical modifications of proteins or peptides. This study explores the hurdles associated with protein and peptide transport, the use of polymeric nanocarriers (both natural and synthetic) to overcome these challenges, and the techniques for fabricating and characterizing nanoparticles.

Emerging Role of Sorting Nexin 17 in Human Health and Disease.

The distortion of the cellular membrane transport pathway has a profound impact on cell dynamics and can drive serious physiological consequences during the process of cell sorting. SNX17 is a member of the Sorting Nexin (SNX) family and plays a crucial role in protein sorting and transport in the endocytic pathway. SNX17, SNX27, and SNX31 belong to the SNX-FERM subfamily and possess the FERM domain, which can assist in endocytic transport and lysosomal degradation. The binding partners of SNX27 have been discovered to number over 100, and SNX27 has been linked to the development of Alzheimer's disease progression, tumorigenesis, cancer progression, and metastasis. However, the role and potential mechanisms of SNX17 in human health and disease remain poorly understood, and the function of SNX17 has not been fully elucidated. In this review, we summarize the structure and basic functions of SNX protein, focusing on providing current evidence of the role and possible mechanism of SNX17 in human neurodegenerative diseases and cardiovascular diseases.

Exploration of the Key Pathways and Genes Involved in Osteoarthritis Genesis: Evidence from Multiple Platforms and Real-World Validation.

Osteoarthritis (OA), a degenerative and chronic joint disease, is essential for identifying novel biomarkers for the clinical diagnosis of OA. We collected 35 OA patients and 32healthy controls from four clinical cohorts and 8 real-world samples from our institute. The activation status of 7530 signalling pathways was calculated via the gene set enrichment analysis (GSEA) algorithm. Ten machine learning algorithms and 101 algorithm combinations were further applied to recognize the most diagnostic genes. KDELR3 was chosen for further validation via immunohistochemical staining to determine its diagnostic value in real-world samples. Sixteen pathways, namely, the cellular respiration chain, protein transport, lysosomal and endocytosis pathways, were activated in OA patients. A total of 101 types of algorithm combinations were considered for the diagnostic model, and 58 were successfully output. The two-step model of glmBoost plus RF had the highest average AUC value of 0.95 and was composed of LY86, SORL1, KDELR3, CSK, PTGS1, and PTGS2. Preferable consistency of the diagnostic mole and real conditions was observed in all four cohorts (GSE55235: Kappa=1.000, P<0.001; GSE55457: Kappa=0.700, P<0.001; GSE82107: Kappa=0.643, P=0.004; GSE1919: Kappa=1.000, P<0.001). KDELR3 was expressed at higher levels in OA patients than were the other genes, and with the help of immunohistochemistry (IHC), we confirmed that OA patients presented high levels of KDELR3 in synovial tissues. The infiltration of immunocytes, macrophages, and natural killer T cells was high in OA patients. KDELR3 might be involved in the activation and infiltration of effector memory CD4 T cells (Rpearson = 0.58, P < 0.001) and natural killer T cells (Rpearson = 0.53, P < 0.001). We constructed and validated a six-gene diagnostic model for OA patients via machine learning, and KDELR3 emerged as a novel biomarker for OA.

Gibberellin and cytokinin signaling antagonistically control female-germline cell specification in Arabidopsis.

How do growth hormones interact to specify female-germline cell types in flowering plants and control production of the first female-germline cell? Here, we find that gibberellin (GA) biosynthesis and signaling are restricted in ovule primordia, with overexpression of receptors and biosynthetic enzymes resulting in multiple and enlarged megaspore mother cells (MMCs) in Arabidopsis. GA signaling machinery interacts with and promotes the degradation of cytokinin (CK) type-B Arabidopsis response regulators (ARR1/10/12), which also directly interact with DELLA proteins. CK biosynthesis and signaling components are expressed in both MMCs and sporophytic cells, with signaling negatively controlled by GA in ovule primordia, and perturbations leading to the induction of multiple, enlarged MMC-like cells. The vacuolar sorting protein SHRUBBY (SHBY) interacts with GA and CK signaling components to block GA-induced degradation. CK signaling restricts multiple sub-epidermal cells in distal ovule primordia from acquiring MMC identity. By balancing degradation activity, GA and CK signaling antagonistically control female-germline cell specification.

SECOND SOUTHERN SCIENCE CONFERENCE: HEALTH SESSIONS

The Second Southern Science Conference brought together international researchers focused on health and diseases. The event featured expert lectures across various fields, including cell biology, neurobiology, immunology, and pharmacology. Notable presentations covered new tools for cell biology research, such as the RUSH and RUCH systems, which enable studying protein trafficking in cells. In microbiology, key topics included antibiotic resistance and bacterial and viral infections. In neurobiology, researchers presented advances in treating neurodegenerative diseases, including the use of nanoparticles for drug delivery. The conference also addressed immunology issues, such as COVID-19 vaccination effects, and aspects of diet in cell physiology. The event concluded with a lecture on academic entrepreneurship, demonstrating how research can be transformed into commercial products. The conference, hosted at the Universidad de Mendoza, provided a unique opportunity for sharing scientific knowledge and advances in healthcare, with the next edition scheduled for 2026.

Impaired chaperone-mediated autophagy leads to abnormal SORT1 (sortilin 1) turnover and CES1-dependent triglyceride hydrolysis

ABSTRACT SORT1 (sortilin 1), a member of the the Vps10 (vacuolar protein sorting 10) family, is involved in hepatic lipid metabolism by regulating very low-density lipoprotein (VLDL) secretion and facilitating the lysosomal degradation of CES1 (carboxylesterase 1), crucial for triglyceride (TG) breakdown in the liver. This study explores whether SORT1 is targeted for degradation by chaperone-mediated autophagy (CMA), a selective protein degradation pathway that directs proteins containing KFERQ-like motifs to lysosomes via LAMP2A (lysosomal-associated membrane protein 2A). Silencing LAMP2A or HSPA8/Hsc70 with siRNA increased cytosolic SORT1 protein levels. Leupeptin treatment induced lysosomal accumulation of SORT1, unaffected by siLAMP2A co-treatment, indicating CMA-dependent degradation. Human SORT1 contains five KFERQ-like motifs (658VVTKQ662, 730VREVK734, 733VKDLK737, 734KDLKK738, and 735DLKKK739), crucial for HSPA8 recognition; mutating any single amino acid within these motifs decreased HSPA8 binding. Furthermore, compromised CMA activity resulted in elevated SORT1-mediated degradation of CES1, contributing to increased lipid accumulation in hepatocytes. Consistent with in vitro findings, LAMP2A knockdown in mice exacerbated high-fructose diet-induced fatty liver, marked by increased SORT1 and decreased CES1 levels. Conversely, LAMP2A overexpression promoted SORT1 degradation and CES1D accumulation, counteracting fasting-induced CES1D suppression through CMA activation. Our findings reveal that SORT1 is a substrate of CMA, highlighting its crucial role in directing CES1 to lysosomes. Consequently, disrupting CMA-mediated SORT1 degradation significantly affects CES1-dependent TG hydrolysis, thereby affecting hepatic lipid homeostasis.

Mechanistic exploration of ubiquitination-mediated pathways in cerebral ischemic injury.

The ubiquitin-proteasome system (UPS) plays a pivotal role in regulating protein homeostasis and cellular processes, including protein degradation, trafficking, DNA repair, and cell signaling. During cerebral ischemia, ischemic conditions profoundly disrupt UPS activity, leading to proteasomal dysfunction and the accumulation of abnormal proteins. This imbalance contributes to neuronal injury and cell death observed in ischemic stroke. The UPS is intricately linked to various signaling pathways crucial for neuronal survival, inflammation, and cellular stress response, such as NF-κB, TRIM, TRIP, JAK-STAT, PI3K/Akt, and ERK1/2. Alterations in the ubiquitination process can significantly impact the activation and regulation of these pathways, exacerbating ischemic brain injury. Therapeutic approaches targeting the UPS in cerebral ischemia aim to rebalance protein levels, reduce proteotoxic stress, and mitigate neuronal injury. Strategies include proteasome inhibition, targeting specific ubiquitin ligases and deubiquitinating enzymes, and modulating ubiquitination-mediated regulation of key signaling pathways implicated in ischemia-induced pathophysiology. Therefore, the present review discusses the molecular mechanisms underlying UPS dysfunction in ischemic stroke is crucial for developing effective therapeutic interventions. Modulating ubiquitination-mediated pathways through therapeutic interventions targeting specific UPS components holds significant promise for mitigating ischemic brain injury and promoting neuroprotection and functional recovery in patients with cerebral ischemia.

Single-Cell Transcriptomic Dataset of RPGR-associated Retinitis Pigmentosa Patient-Derived Retinal Organoids

X-linked retinitis pigmentosa (XLRP) is a severe hereditary retinal disorder marked by progressive vision loss due to photoreceptor dysfunction. The retinitis pigmentosa GTPase regulator (RPGR) gene, responsible for most XLRP cases, encodes a protein crucial for the transport of visual signal proteins between the photoreceptor inner and outer segments. However, the mechanism of RPGR mutation causing photoreceptor disorder is not clear and effective treatments remain elusive. This study utilized retinal organoids (ROs) derived from normal and RPGR-mutant human induced pluripotent stem cells (hiPSC) at four developmental stages (40, 90, 150, and 200 days). Single-cell RNA sequencing (scRNA-seq) was conducted on 71,096 cells, including 33,839 cells from the control group and 37,257 cells from the RPGR group. Key retinal cell types were identified and the obtained scRNAseq dataset was validated reliable and high -quality. This study has provided data resources and references for exploring the mechanism of RPGR-related retinal degeneration and support the development of targeted therapies.

GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway

Glycosylation, which plays an important role in modifying lipids and sorting of proteins, is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking LYSET/TMEM251, a retention factor for Golgi N-acetylglucosamine-1-phosphotransferase (GNPT), display SPPL3-dependent hypersecretion of the Golgi membrane protein B4GALT5. We demonstrate that in wild-type cells B4GALT5 is tagged with mannose 6-phosphate (M6P), a sorting tag typical of soluble lysosomal hydrolases. Hence, M6P-tagging of B4GALT5 may represent a novel degradative lysosomal pathway. We also observed B4GALT5 hypersecretion and prominent destabilization of LYSET–GNPT complexes, impaired M6P-tagging, and disturbed maturation and trafficking of lysosomal enzymes in multiple human cell lines lacking the COPI adaptors GOLPH3 and GOLPH3L. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. Thus, by ensuring the cis-Golgi localization of the LYSET–GNPT complex and maintaining its Golgi polarity, GOLPH3/GOLPH3L is essential for the integrity of the M6P-tagging machinery and homeostasis of lysosomes.