Endocytosis Of Proteins Research Articles

Endocytosis mediated by megalin and cubilin is involved in enamel development.

Endocytosis of enamel matrix proteins (EMPs) by ameloblasts is a key process in the mineralization of enamel during the maturation stage of amelogenesis. However, the relevant receptor mediating endocytosis of EMPs is still unclear. The aim of this study was to explore potential endocytic receptors involved in this process. Two endocytic receptors, megalin, and cubilin, were found to be distributed in ameloblasts of mouse incisors and molars during the secretory and maturation stages. Megalin was located at the distal end of ameloblasts during the maturation stage when proteolysis and recycling were the most active. Megalin and cubilin were also expressed in an ameloblast-lineage cell (ALC) line. The immunoelectron microscopy results showed that megalin was positively labeled on the vesicle structures of ALC, where endocytosis happened. Immunofluorescence showed that megalin and cubilin were colocalized with amelogenin, and the absorption of amelogenin was significantly reduced when megalin and cubilin were inhibited by their inhibitor, receptor-associated protein (RAP). Knockdown of megalin and cubilin with siRNA also reduced the ability of ALC to absorb amelogenin. The results of this study suggest that megalin and cubilin are involved in the absorption process of ameloblasts during amelogenesis.

Adsorption Force of Fibronectin Regulates Protein Reorganization, Desorption, and Endocytosis.

Protein adsorption on biomaterials occurs before cell adhesion. To adapt the properties of biomaterials, adhered cells may utilize and modify adsorbed proteins for survival and function. In this process, the protein-material interfacial force (Fad) is supposed to play vital roles, which, however, has received little attention. Here, we found that rat mesenchymal stem cells (rMSCs) can utilize the adsorbed fibronectin (FN) via reorganization, desorption, or endocytosis, and these utilization processes are regulated by Fad through regulating cell adhesion and force balance between the cell traction force and Fad. Furthermore, protein utilization has an Fad-dependent temporal sequence. On low Fad surface, FN endocytosis might happen prior to FN desorption and aggregation. This work confirms the importance of Fad in protein utilization and provides new insight into the mechanism by which cells process their surrounding ECM proteins, which may help to guide the design of better biomaterials.

Renal nerves and hypertension contribute to impaired proximal tubule megalin-mediated albumin uptake in renovascular hypertensive rats.

Proteinuria, especially albuminuria, serves as an independent risk factor for progression in cardiovascular and renal diseases. Clinical and experimental studies have demonstrated that renal nerves contribute to renal dysfunction in arterial hypertension (AH). This study hypothesizes that renal nerves mediate the mechanisms of protein endocytosis by proximal tubule epithelial cells (PTEC) and glomerular function; with dysregulation of the renal nerves contributing to proteinuria in Wistar rats with renovascular hypertension (2-kidney, 1-clip model, 2K-1C). Reduced albumin uptake and increased internalization of endocytic receptor megalin in PTEC were found in both the clipped and contralateral kidneys of 2K-1C rats. Renal denervation (DNx) or hydralazine treatment restored these parameters. Moreover, DNx, but not hydralazine, reduced serum creatinine and recovered podocyte numbers in the contralateral kidney of 2K-1C rats. Thus, our data suggest that renal nerves and high arterial pressure contribute to decreased albumin reabsorption by cellular redistribution of megalin in PTEC, while renal nerves remarkably drive glomerular dysfunction in renovascular hypertension, independently of their effect on blood pressure. Created with BioRender.com.

Single-Cell RNA-Seq Reveals the Pseudo-temporal Dynamic Evolution Characteristics of ADSCs to Neuronal Differentiation

Adipose-derived stromal cells (ADSCs) are commonly used in regenerative medicine, but the genetic features of their development into neuronal cells are unknown. This study used single-cell RNA sequencing (scRNA-seq) to reveal gene expression changes during ADSCs to neuronal differentiation. Sequencing of the ADSCs group, the prei-1d group, and the induction 1 h, 3 h, 5 h, 6 h, and 8 h groups was performed using the BD Rhapsody platform. Sequence data were analyzed using t-SNE, Monocle2, GO, and KEGG algorithms. Results showed that a total of 38,453 cells were collected, which were divided into 0–13 clusters. Monocle2 structured analysis revealed that ADSCs were located at the beginning of the trajectory, and the cells after 5 h of induction were mainly distributed at the end of the trajectory in branches 1 and 2. Up-regulated differentially expressed genes (DEGs) at 5 h after induction enriched GO items including cellular protein metabolism, cell adhesion, endocytosis, and cell migration. KEGG analysis showed that induced 6 h and 8 h groups mainly enriched pathways were oxidative phosphorylation, glutathione metabolism, and expression of Parkinson's disease-related genes. In conclusion, two distinct cell state mechanisms stimulate ADSCs to develop into mature neurons. ADSCs induced for 5 h had developed into mature neurons. Later, the differentiated cells undergo degenerative changes associated with senescence.

Programmed cell death-related ABI1 is a critical mediator of abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by localized dilation of the abdominal aorta, posing a significant risk of rupture and fatal hemorrhage. While surgical and endovascular repair techniques have advanced, the underlying mechanisms driving AAA development remain unclear, hindering the development of effective preventive and therapeutic strategies. Using bioinformatics analysis of publicly available data sets, the study identified a strong correlation between cell death (CD) score and different types of programmed cell death scores in AAA samples. WGCNA analysis revealed a module enriched in genes related to proteasome-mediated protein degradation, nuclear envelope, and endocytosis, significantly correlated with CD score. Further analysis identified ABI1 as a dominant feature gene, highlighting its potential role in AAA pathogenesis. In vitro validation using an Angiotensin II-induced AAA model in human aortic smooth muscle cells demonstrated that siRNA-mediated knockdown of ABI1 significantly reduced cell apoptosis, migration, and the expression of pro-apoptotic proteins, confirming ABI1's crucial role in promoting CD and AAA progression. The findings suggest that ABI1 may represent a promising therapeutic target for the prevention and treatment of AAA. Further research is warranted to fully understand the role of ABI1 in AAA and to develop targeted therapies based on this promising target.

Effect of hypernatremia on protein reabsorption in renal proximal tubules of the lake frog <I>Pelophylax ridibundus</I>

Protein reabsorption in the kidney proximal tubules occurs simultaneously with the transport of ions and water, but little is known about the dependence of receptor-mediated protein endocytosis on water-salt balance changes. The aim of the study was to investigate tubular reabsorption and intracellular vesicular transport of various proteins in a model of hypernatremia in lake frogs (Pelophylax ridibundus). Frogs were injected with hypertonic sodium chloride solution (0.75 M NaCl) 1 hour before injection of green or yellow fluorescent proteins (GFP or YFP), as well as lysozyme. The method of fluorescent immunohistochemistry was used for detection of lysozyme and endocytic receptor megalin in kidney sections. Specimens were investigated using laser scanning confocal microscopy. The intensity of fluorescent signals of proteins and megalin in proximal tubular cells was determined on the images obtained. To study the dynamics of endocytosis, an automated method for quantifying colocalized protein and megalin signals was used. A statistically significant decrease in the reabsorption of GFP, YFP and lysozyme in the proximal tubules after 0.75 M of NaCl injection was found. The accumulation of proteins in the early endocytic compartment and decrease in their entry into late endosomes and lysosomes are shown, that is considered as evidence of a delay in intracellular vesicular transport in hypernatremia. The data obtained were analyzed in connection with changes in blood parameters and kidney activity during osmoregulation, and also with the role of chloride channels in receptor-mediated protein endocytosis. It can be assumed that increased ion transport in the proximal tubules cells in hypernatremia leads to decreased reabsorption capacity of epitheliocytes and delayed intracellular transport of proteins.

Programmable Circular Multivalent Nanobody‐Targeting Chimeras (mNbTACs) for Multireceptor‐Mediated Protein Degradation and Targeted Drug Delivery

AbstractMultispecific therapeutics hold significant promise in drug delivery, protein degradation, and cell recruitment to address clinical issues of tumor heterogeneity, resistance, and immune evasion. However, their modular engineering remains challenging. We developed a targeted degradation platform, termed multivalent nanobody‐targeting chimeras (mNbTACs), by encoding diverse nanobody codons on a circular template using DNA printing technology. The homo‐ or hetero‐ mNbTACs specifically recognized membrane targets in a multivalent manner and simultaneously recruited scavenger receptors to favor clathrin‐/caveolae‐dependent endocytosis and lysosomal degradation of multiple proteins with high efficiency and selectivity. We demonstrated that a bispecific doxorubicin‐loaded mNbTAC, named Doxo‐mvNbsPPH, passively accumulated at tumor sites, specifically interacted with PD−L1 and HER2 targets, and was rapidly transported into lysosome, inducing potent immunogenic cell death and alleviating immune checkpoint evasion. The synergistic boosting of innate and adaptive immunity promoted the infiltration and proliferation of CD8+ T cells in tumor microenvironment (an 11‐fold increase) with high toxicity and low exhaustion, eventually enhancing antitumor efficacy. Our mNbTAC platform provides multispecific therapeutics with variable valences and programmed species, whereas it induces targeted protein degradation through multireceptor‐mediated endocytosis and lysosomal degradation without the need for lysosome‐targeting receptors, representing a general and modular tool to harness extracellular proteome for disease treatment.

Programmable Circular Multivalent Nanobody-Targeting Chimeras (mNbTACs) for Multireceptor-Mediated Protein Degradation and Targeted Drug Delivery.

Multispecific therapeutics hold significant promise in drug delivery, protein degradation, and cell recruitment to address clinical issues of tumor heterogeneity, resistance, and immune evasion. However, their modular engineering remains challenging. We developed a targeted degradation platform, termed multivalent nanobody-targeting chimeras (mNbTACs), by encoding diverse nanobody codons on a circular template using DNA printing technology. The homo- or hetero- mNbTACs specifically recognized membrane targets in a multivalent manner and simultaneously recruited scavenger receptors to favor clathrin-/caveolae-dependent endocytosis and lysosomal degradation of multiple proteins with high efficiency and selectivity. We demonstrated that a bispecific doxorubicin-loaded mNbTAC, named Doxo-mvNbsPPH, passively accumulated at tumor sites, specifically interacted with PD-L1 and HER2 targets, and was rapidly transported into lysosome, inducing potent immunogenic cell death and alleviating immune checkpoint evasion. The synergistic boosting of innate and adaptive immunity promoted the infiltration and proliferation of CD8+ T cells in tumor microenvironment (an 11-fold increase) with high toxicity and low exhaustion, eventually enhancing antitumor efficacy. Our mNbTAC platform provides multispecific therapeutics with variable valences and programmed species, whereas it induces targeted protein degradation through multireceptor-mediated endocytosis and lysosomal degradation without the need for lysosome-targeting receptors, representing a general and modular tool to harness extracellular proteome for disease treatment.

Discovery and evaluation of HW161023 as a potent and orally active AAK1 inhibitor

AAK1, also known as AP2-associated protein kinase 1, is an enzyme that belongs to the family of serine/threonine protein kinases. It regulates the assembly and disassembly of clathrin-coated pits and thereby protein endocytosis, by phosphorylating the μ2 subunit of the AP2 complex, which is a key component of clathrin-coated vesicles. LX9211 is currently the only selective small molecule AAK1 inhibitor at the clinical trial stage for diabetic peripheral neuropathic pain, which was found to be safe and well tolerated in healthy participants in phase I clinical trials. The present manuscript described a series of fused-ring derivatives as a novel class of potent AAK1 inhibitors, resulting in the discovery of compound 5, namely HW161023, which showed high inhibitory potency against AAK1 enzyme and satisfactory oral pharmacokinetic profile with weaker HepG2 cell toxicity and hERG inhibition than LX9211.

Novel TBC1D8B variant causes neonatal nephrotic syndrome combined with acute kidney injury

BackgroundNephrotic syndrome (NPHS), characterized by proteinuria, hypoalbuminemia, and edema, can be caused by genetic variations. TBC1D8B was recently discovered as a novel disease-causing gene for X-linked NPHS. With only a few reported cases, the clinical manifestations associated with variants of this gene need to be further examined.MethodsWe recruited a newborn with NPHS complicated by acute kidney injury (AKI) and his parents and tested the potential genetic cause of the disease through trio-whole exome sequencing and Sanger sequencing. Western blotting (WB) was performed using a mutant plasmid to evaluate mutant protein expression levels. Since the TBC1D8B protein interacts with RAB proteins to catalyze the GTPase hydrolysis process, immunofluorescence (IF) can be used to verify the interaction between the TBC1D8B mutant protein and RAB11A/RAB11B, and thus to confirm its effect on the endocytosis and vesicle recycling functions of RAB proteins within the cell.ResultsThe child, at 1 month, showed severe edema and proteinuria and unexplained coma with epilepsy. Ultrasound examination revealed multiple organ enlargement, and MRI showed nonspecific high diffusion-weighted imaging signal characteristics in the splenium of the corpus callosum. Hematoxylin and eosin staining showed diffuse inflammatory cell infiltration in the renal interstitium and multifocal renal tubule lumen expansion. Diffuse fusion of podocyte foot processes was observed under electron microscopy, indicating glomerular podocyte lesions. Genetic testing revealed a maternally inherited novel hemizygous variant, NM_017752: c.628 A > T, p.Lys210Ter, in TBC1D8B. In vitro functional experiments showed that this variant may lead to TBC1D8B protein degradation. IF results showed disrupted interaction with RAB11A/RAB11B, that then affects the biological function of RAB proteins in the process of cell intimal vesicle formation and intracellular transport.ConclusionThis study will enrich the mutational and phenotypic spectra of TBC1D8B and demonstrate the potential of this gene variants to cause early-onset NPHS leading to severe kidney disease.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

A comparative study on the toxic effects of lead pollution and nanoplastic-lead mixed pollution on red drum and their detoxification strategies

Nanoplastics and heavy metals pose various adverse effects on marine organisms. However, the combined toxicity of nanoplastics and lead pollution to marine fish is not fully understood. This study investigates the toxic effects and detoxification strategies of lead pollution (p07) compared to nanoplastic-lead mixed pollution (m07) in red drum during exposure and recovery phases. Under m07 pollution, the maximum lead content in muscle was 22.61 mg/kg, which was significantly higher than the 15.82 mg/kg observed under p07 pollution. This finding demonstrated that nanoplastics significantly enhance lead accumulation, leading to more severe toxic effects on red drum. Histological analyses revealed that lipid droplets in the liver and epithelial lifting in the gills were the primary lesion types. During the exposure periods, red drum primarily detoxified lead through cellular renewal and the removal of damaged proteins under p07 pollution. Conversely, under m07 pollution, detoxification relied on cellular senescence, apoptosis, endocytosis, and the removal of damaged proteins. In the recovery phases, red drum predominantly recovered through cell proliferation and antioxidant responses under p07 pollution. Under m07 pollution, the focus shifted to functional protein synthesis, apoptosis, endocytosis, and lipid metabolism. This study offers valuable insights into the monitoring and management of combined environmental pollution.

A high cholesterol diet aggravates experimental colitis through SREBP2-modulated endocytosis and degradation of occludin and Zo-1 proteins.

Disrupted cholesterol homeostasis plays a critical role in the development of multiple diseases, such as cardiovascular disease and cancer. However, the role of cholesterol in inflammatory bowel disease (IBD) remains unclear. In the present study, we investigated whether and how high levels of cholesterol in the diet affect experimental colitis in mice. A normal diet supplemented with 1.25% cholesterol (high cholesterol diet) caused more severe colitis and aggravated the disruption of intestinal tight junction structure, accompanied by higher colonic tissue total cholesterol (TC) levels in a dextran sulfate sodium (DSS)-induced experimental colitis mouse model. Cholesterol aggravated DSS-induced intestinal epithelial barrier impairment and nuclear sterol regulatory element-binding protein 2 (nSREBP2) inhibition both in vivo and in vitro. In addition, nSREBP2 overexpression ameliorated cholesterol-induced intestinal epithelial barrier disruption in Caco2 cells. Interestingly, inhibition of SREBP2 disrupted intestinal epithelial barrier in the absence of cholesterol. Furthermore, SREBP2 regulated the protein expression of tight junction proteins (occludin/Zo-1) via modulating caveolin-1-mediated endocytosis and lysosomal degradation. Analysis of UK Biobank data indicated that, in fully adjusted models, higher serum TC concentrations were an independent protective factor for IBD incidence. The sterol regulatory element-binding factor 2 (SREBF2) gene rs2228313 (G/C) genetic variant was associated with the incidence of IBD and the CC genotype of SREBF2 rs2228313 was associated with higher serum TC levels and decreased the risk of IBD. In summary, a high cholesterol diet aggravates DSS-induced colitis in mice by down-regulating nSREBP2 expression, thereby promoting the endocytic degradation of tight junction proteins. In humans, SREBF2 gene single nucleotide polymorphism rs2228313 and serum TC levels are associated with IBD incidence.

Selenium Treatment Ameliorates the Adverse Effects Caused by Dynamin Gene Knockdown in Bombyx mori.

Our previous research reported the influence of 50 μM selenium (Se) on the cytosolization (endocytosis) pathway, which in turn stimulates the growth and development of Bombyx mori. Lately, dynamin is recognized as one of the key proteins in endocytosis. To explore the underlying mechanisms of Se impact, the dynamin gene was knocked down by injecting siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3). This was followed by an analysis of the target gene and levels of silk protein genes, as well as growth and developmental indices, Se-enrichment capacity, degree of oxidative damage, and antioxidant capacity of B. mori. Our findings showed a considerable decrease in the relative expression of the dynamin gene in all tissues 24 h after the interference and a dramatic decrease in the silkworm body after 48 h. RNAi dynamin gene decreased the silkworm body weight, cocoon shell weight, and the ratio of cocoon. In the meantime, malondialdehyde level increased and glutathione level and superoxide dismutase/catalase activities decreased. 50 μM Se markedly ameliorated these growth and physiological deficits as well as decreases in dynamin gene expression. On the other hand, there were no significant effects on fertility (including produced eggs and laid eggs) between the interference and Se treatments. Additionally, the Se content in the B. mori increased after the dynamin gene interference. The dynamin gene was highly expressed in the silk gland and declined significantly after interference. Among the three siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3), the dynamin-2 displayed the highest interference effects to target gene expression. Our results demonstrated that 50 μM Se was effective to prevent any adverse effects caused by dynamin knockdown in silkworms. This provides practical implications for B. mori breeding industry.

Accumulation of dually targeted StGPT1 in chloroplasts mediated by StRFP1, an E3 ubiquitin ligase, enhances plant immunity.

Chloroplasts play a crucial role in essential processes, such as photosynthesis and the synthesis of primary and diverse secondary metabolites. Recent studies have also highlighted their significance linked to phytohormone production in plant immunity, especially SA and JA. Ubiquitination, a key posttranslational modification, usually leads to target protein degradation, which acts as a signal for remodeling the proteome via the induction of protein endocytosis or targeting to other membrane associated systems. Previously, the potato E3 ligase StRFP1 was shown to enhance resistance against Phytophthora infestans, but its mechanism remained unclear. Here, we demonstrate that StRFP1 interacted with the dually localized plastid glucose 6-phosphate transporter StGPT1 on the endoplasmic reticulum (ER). Transiently expressed StGPT1-GFP located on the chloroplast and ER in plant cells. Overexpression of StGPT1 enhances late blight resistance in potato and Nicotiana benthamiana, activates immune responses, including ROS bursts and up-regulation of PTI marker genes. The resistance function of StGPT1 seems to be related to its dual localization. Remarkably, StRFP1 ubiquitinates StGPT1 at the ER, possibly due to its merely transient function in peroxisomes, leading to apparent accumulation in chloroplasts. Our findings point to a novel mechanism by which a plant E3 ligase contributes to immunity via interacting with dually targeted GPT1 at the ER of plant cells.

A novel transgenic mouse model highlights molecular disruptions involved in the pathogenesis of Dent disease 1

Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl-/H+exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at exploring therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease.

The C-terminal proline-rich repeats of Enteropathogenic E. coli effector EspF are sufficient for the depletion of tight junction membrane proteins and interactions with early and recycling endosomes

BackgroundEnteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats.ResultsWe generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11.ConclusionsOur study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.

ACLS4 could be a potential therapeutic target for severe acute pancreatitis

Acute pancreatitis (AP) is currently among the most prevalent digestive diseases. The pathogenesis of AP remains elusive, and there is no specific treatment. Therefore, identifying novel therapeutic targets is imperative for effective management and prevention of AP. In this study, we conducted a comprehensive transcriptomic analysis of peripheral blood from patients with AP and the pancreatic tissue from a mouse model of AP. Our analyses revealed that mouse model of AP exhibited a higher enrichment of mitogen-activated protein kinase signaling, endocytosis, apoptosis and tight junction pathways than the control. Subsequent weighted gene co-expression network analysis identified 15 gene modules, containing between 50 and 1000 genes each, which demonstrated significant correlations within samples from patients with AP. Further screening identified four genes (ACSL4, GALNT3, WSB1, and IL1R1) that were significantly upregulated in severe acute pancreatitis (SAP) in both human and mouse samples. In mouse models of SAP, ACSL4 was significantly upregulated in the pancreas, whereas GALNT3, WSB1, and IL1R1 were not. Lastly, we found that a commercially available ACSL4 inhibitor, PRGL493, markedly reduced IL-6 and TNFα expression, alleviated pancreatic edema and necrosis, and diminished the infiltration of inflammatory cells. In conclusion, this study comprehensively depicts the key genes and signaling pathways implicated in AP and suggests the potential of ACSL4 as a novel therapeutic target for SAP. These findings provide valuable insights for further exploration of therapeutic strategies for SAP.