IU Indianapolis ScholarWorks :: Browsing by Subject "Endocytosis"

Browsing by Subject "Endocytosis"

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Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications
(American Physiological Society, 2022) Molitoris, Bruce A.; Sandoval, Ruben M.; Yadav, Shiv Pratap S.; Wagner, Mark C.; Medicine, School of Medicine
For nearly 50 years the proximal tubule (PT) has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes. Several genetic diseases, nonselective PT cell defects, chronic kidney disease (CKD), and acute PT injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PT injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism, and transcytosis are being reexamined with the use of techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multireceptor complex that mediates albumin binding and uptake and directs proteins for lysosomal degradation after endocytosis. Albumin transcytosis is mediated by a pH-dependent binding affinity to the neonatal Fc receptor (FcRn) in the endosomal compartments. This reclamation pathway rescues albumin from urinary losses and cellular catabolism, extending its serum half-life. Albumin that has been altered by oxidation, glycation, or carbamylation or because of other bound ligands that do not bind to FcRn traffics to the lysosome. This molecular sorting mechanism reclaims physiological albumin and eliminates potentially toxic albumin. The clinical importance of PT albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussion of genetic disorders and therapeutic considerations.
Dynamic vs Static ABCG2 Inhibitors to Sensitize Drug Resistant Cancer Cells
(Public Library of Science, 2010-12-07) Peng, Hui; Qi, Jing; Dong, Zizheng; Zhang, Jian-Ting; Pharmacology and Toxicology, School of Medicine
Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in multidrug resistance and protecting cancer stem cells. ABCG2-knockout had no apparent adverse effect on the development, biochemistry, and life of mice. Thus, ABCG2 is an interesting and promising target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and for eliminating cancer stem cells. Previously, we reported a novel two mode-acting ABCG2 inhibitor, PZ-39, that induces ABCG2 degradation in addition to inhibiting its activity. In this manuscript, we report our recent progresses in identifying two different groups of ABCG2 inhibitors with one inhibiting only ABCG2 function (static) and the other induces ABCG2 degradation in lysosome in addition to inhibiting its function (dynamic). Thus, the inhibitor-induced ABCG2 degradation may be more common than we previously anticipated and further investigation of the dynamic inhibitors that induce ABCG2 degradation may provide a more effective way of sensitizing ABCG2-mediated MDR in cancer chemotherapy.
Exploring Class‐II PI3K Inhibition for the treatment of Alzheimer’s Disease: Virtual Screening for PI3KC2A Inhibitors
(Wiley, 2025-01-09) Jadala, Chetna; Robo, Michael T.; Richardson, Timothy I.; Pharmacology and Toxicology, School of Medicine
Background: Focusing on novel AD treatments, the TREAT‐AD centers offer an array of free research tools, shared via the AD Knowledge Portal in a Target Enablement Package (TEP). This abstract showcases the research conducted by the IUSM‐Purdue TREAT‐AD Center, specifically focusing on Targeting class‐II PI3K’s as a potential breakthrough in AD therapy. Endocytosis within the brain encompasses diverse pathways for internalizing extracellular cargoes and receptors into cells. The prominent routes include clathrin‐mediated endocytosis and phagocytosis. Endocytosis plays a crucial role in processing amyloid precursor protein (APP) leading to abnormal production of Aβ peptides. Recycling endosomes are vital for delivering and eventually releasing Aβ into the brain. Recent research emphasizes the pivotal role of PI3K‐C2α, a class II PI3K member, in regulated endocytosis through its clathrin‐binding domain. Its localization spans clathrin‐coated pits, endocytic vesicles, early endosomes, and the trans‐Golgi network, generating phosphatidylinositol 3‐phosphate (PtdIns(3)P) and/or phosphatidylinositol 3,4‐bisphosphates (PtdIns(3,4)P2) in vivo. Targeting clathrin‐mediated endocytosis by inhibiting PI3K‐C2α, a key regulator in clathrin coated vesicle formation, could be a potential therapeutic strategy against Alzheimer’s disease. Method: We conducted extensive virtual screenings of vast compound libraries to determine potent small molecules inhibiting PI3K‐C2α. Employing shape‐based screening, and clustering techniques, we identified leading compounds for subsequent in vitro kinase assays. Compounds exhibiting nanomolar activity were selected for further investigation. Leveraging these findings, we conducted Structure‐Activity Relationship (SAR) studies, optimizing analogs to enhance binding affinity and cellular pharmacology. Result: We have identified novel PI3K‐C2α inhibitors and are in the initial stages of optimization. These compounds exhibit promising target engagement, pending further assessment for biochemical activity and cellular pharmacology. In silico assessments suggest their structures are ideal for CNS drug discovery plans. Conclusion: Inhibiting PI3K‐C2α stands as a promising therapeutic approach for Alzheimer’s disease. We've discovered unique molecular structures that inhibit the enzyme. Our findings suggest potential probe molecules for validating the target and developing lead compounds for clinical investigations.
hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation
(Elsevier B.V., 2008-03) Ren, Yongming; Cheng, Long; Rong, Zhili; Li, Zhiyong; Li, Yinghua; Zhang, Xinjun; Xiong, Shiqin; Hu, Jim; Fu, Xin-Yuan; Chang, Zhijie; Department of Microbiology & Immunology, IU School of Medicine
Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.
Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology
(Elsevier, 2017-09-01) Sandoval, Ruben M.; Molitoris, Bruce A.; Medicine, School of Medicine
The kidney is a complex and dynamic organ with over 40 cell types, and tremendous structural and functional diversity. Intravital multi-photon microscopy, development of fluorescent probes and innovative software, have rapidly advanced the study of intracellular and intercellular processes within the kidney. Researchers can quantify the distribution, behavior, and dynamic interactions of up to four labeled chemical probes and proteins simultaneously and repeatedly in four dimensions (time), with subcellular resolution in near real time. Thus, multi-photon microscopy has greatly extended our ability to investigate cell biology intravitally, at cellular and subcellular resolutions. Therefore, the purpose of the chapter is to demonstrate how the use in intravital multi-photon microscopy has advanced the understanding of both the physiology and pathophysiology of the kidney.
Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics
(Frontiers Media, 2022-03-24) Molitoris, Bruce A.; Sandoval, Ruben M.; Wagner, Mark C.; Medicine, School of Medicine
Intravital multiphoton microscopy has empowered investigators to study dynamic cell and subcellular processes in vivo within normal and disease organs. Advances in hardware, software, optics, transgenics and fluorescent probe design and development have enabled new quantitative approaches to create a disruptive technology pioneering advances in understanding of normal biology, disease pathophysiology and therapies. Offering superior spatial and temporal resolution with high sensitivity, investigators can follow multiple processes simultaneously and observe complex interactions between different cell types, intracellular organelles, proteins and track molecules for cellular uptake, intracellular trafficking, and metabolism in a cell specific fashion. The technique has been utilized in the kidney to quantify multiple dynamic processes including capillary flow, permeability, glomerular function, proximal tubule processes and determine the effects of diseases and therapeutic mechanisms. Limitations include the depth of tissue penetration with loss of sensitivity and resolution due to scattered emitted light. Tissue clearing technology has virtually eliminated penetration issues for fixed tissue studies. Use of multiphoton microscopy in preclinical animal models offers distinct advantages resulting in new insights into physiologic processes and the pathophysiology and treatment of diseases.
Lrpap1 (RAP) Inhibits Proximal Tubule Clathrin Mediated and Clathrin Independent Endocytosis, Ameliorating Renal Aminoglycoside Nephrotoxicity
(Wolters Kluwer, 2023) Wagner, Mark C.; Sandoval, Ruben M.; Yadav, Shiv Pratap S.; Campos, Silvia B.; Rhodes, George J.; Phillips, Carrie L.; Molitoris, Bruce A.; Medicine, School of Medicine
Background: Proximal tubules (PTs) are exposed to many exogenous and endogenous nephrotoxins that pass through the glomerular filter. This includes many small molecules, such as aminoglycoside and myeloma light chains. These filtered molecules are rapidly endocytosed by the PTs and lead to nephrotoxicity. Methods: To investigate whether inhibition of PT uptake of filtered toxins can reduce toxicity, we evaluated the ability of Lrpap1 or receptor-associated protein (RAP) to prevent PT endocytosis. Munich Wistar Frömter rats were used since both glomerular filtration and PT uptake can be visualized and quantified. The injury model chosen was the well-established gentamicin-induced toxicity, which leads to significant reductions in GFR and serum creatinine increases. CKD was induced with a right uninephrectomy and left 40-minute pedicle clamp. Rats had 8 weeks to recover and to stabilize GFR and proteinuria. Multiphoton microscopy was used to evaluate endocytosis in vivo and serum creatinine, and 24-hour creatinine clearances were used to evaluate kidney functional changes. Results: Studies showed that preadministration of RAP significantly inhibited both albumin and dextran endocytosis in outer cortical PTs. Importantly, this inhibition was found to be rapidly reversible with time. RAP was also found to be an excellent inhibitor of PT gentamicin endocytosis. Finally, gentamicin administration for 6 days resulted in significant elevation of serum creatinine in vehicle-treated rats, but not in those receiving daily infusion of RAP before gentamicin. Conclusions: This study provides a model for the potential use of RAP to prevent, in a reversible manner, PT endocytosis of potential nephrotoxins, thus protecting the kidney from damage.
Pou5f1-dependent EGF expression controls E-cad endocytosis, cell adhesion, and zebrafish epiboly movements
(Elsevier, 2013) Song, Sungmin; Eckerle, Stephanie; Onichtchouk, Daria; Marrs, James A.; Nitschke, Roland; Driever, Wolfgang; Biology, School of Science
Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that, in zebrafish maternal and zygotic (MZ)spg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cadherin (E-cad) endosomal trafficking, and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGF receptor may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other.
The role of acid sphingomyelinase in autophagy
(2014-07-11) Justice, Matthew Jose; Petrache, Irina; Blum, Janice Sherry, 1957-; Wek, Ronald C.
Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.
TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells
(American Society for Cell Biology, 2014-11-15) Gallo, Luciana I.; Ruiz, Wily G.; Clayton, Dennis R.; Liu, Yong-Jian; Jiang, Yu; Fukuda, Mitsunori; Apodaca, Gerard; Yin, Xiao-Ming; Department of Pathology & Laboratory Medicine, IU School of Medicine
Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the guanine nucleotide-exchange factors and GTPase-activating proteins (GAPs) that regulate its GTP-GDP cycle. We observed that in the presence of Mg(2+) (2.5 mM), TBC1D9B interacted via its Tre2-Bub2-Cdc16 (TBC) domain with Rab11a, Rab11b, and Rab4a in a nucleotide-dependent manner. However, only Rab11a was a substrate for TBC1D9B-stimulated GTP hydrolysis. At limiting Mg(2+) concentrations (<0.5 mM), Rab8a was an additional substrate for this GAP. In polarized Madin-Darby canine kidney cells, endogenous TBC1D9B colocalized with Rab11a-positive recycling endosomes but less so with EEA1-positive early endosomes, transferrin-positive recycling endosomes, or late endosomes. Overexpression of TBC1D9B, but not an inactive mutant, decreased the rate of basolateral-to-apical IgA transcytosis--a Rab11a-dependent pathway--and shRNA-mediated depletion of TBC1D9B increased the rate of this process. In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor. Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A. We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

Browsing by Subject "Endocytosis"

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