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The Renin-Angiotensin System
Published in Austin E. Doyle, Frederick A. O. Mendelsohn, Trefor O. Morgan, Pharmacological and Therapeutic Aspects of Hypertension, 2020
The mechanism of the selective proteolysis of the leucyl-leucine bond of natural and synthetic peptide substrates by renin is not yet fully understood. Experiments using specific inactivators of proteases.41,42 indicate that renin is neither a serine-, cysteine-, or metallo-protease, The discovery that pepstatin, which is an inhibitor of pepsin, cath-epsin D, and other acidic protesases, is also a potent inhibitor of renin,43,44 suggests that renin may belong to this class of enzymes. Interestingly, both of the acid proteases pepsin45 and cathepsin D46 are also capable of hydrolyzing the leucyl-leucine bond of natural45 and synthetic46 renin substrates. This means that care is needed in identifying tissue isorenins since these enzymes may interfere with many renin assays. Further evidence that renin has structural similarities to the acid proteases was the finding that it was inactivated by a diazo compound whose action is relatively specific for this group of enzymes.47 Definitive evidence concerning the amino acid sequence of the active site of renin may be possible now that highly purified preparations of the enzyme are available33,37 by using suitably designed “suicide substrates”48 to label the active site.
Understanding the Interaction of Nanoparticles at the Cellular Interface
Published in D. Sakthi Kumar, Aswathy Ravindran Girija, Bionanotechnology in Cancer, 2023
Fick’s law states that the flux or rate at which a molecule diffuses across the plasma membrane is proportional to the concentration gradient, the membrane surface area, and the molecule’s permeability coefficient [15]. Many molecules have a low permeability coefficient, and they tend to diffuse slowly. However, these molecules are rapidly transported across the membrane by specific transmembrane proteins as a facilitated diffusion, which is a far part of the Fick’s law of diffusion. Small NPs, less than 1 nm, are internalized by diffusion. The entry of engineered NPs into the cell may fall into different categories, as shown in Figure 2.1, based on the size and surface functionalities [16]. Most of the larger particles are internalized either by phagocytosis or micropinocytosis pathways, which are independent of clathrin and caveolin pathways. Phagocytosis or micropinocytosis pathways start membrane protrusions due to actin polymerization, followed by the engulfing of substance to be internalized into the cell. In receptor-mediated endocytosis, there is always a highly specific uptake of NPs with the help of functionalized ligands on the surface of NPs. It is observed that NPs rotate during the internalization process with the diffusion of receptors as a prolog to the wrapping mechanism exhibited by membranes during receptor-mediated endocytosis [17]. Clathrin-mediated endocytosis has been identified as a cellular entry mechanism to internalize specific molecules into the cells [18]. Clathrin is a particular protein coated at the intracellular face of the plasma membrane, which further forms a pit for internalizing specific molecules or engineered NPs in the presence of adaptor and accessory proteins [19]. There is a cascade of signals that are activated during the contact of NPs on the cell surface. Epsin, one of the known adaptor proteins, induces membrane curvature on the cell. That will be followed by the deep invagination by an accessory protein called dynamin, which will help NPs enter the cell. Generally, viruses are considered as a naturally made nanoparticle, and they follow a variety of endocytic pathways, based on their structure, for the entry into a mammalian cell [20]. Several chemical inhibitors can be used as a tool for direct probing of endocytic events in live cells [21].
Notch signaling in spermatogenesis and male (in)fertility
Published in Rajender Singh, Molecular Signaling in Spermatogenesis and Male Infertility, 2019
Mahitha Sahadevan, Pradeep G. Kumar
In the canonical notch pathway, the notch signal is initiated by the interaction of the single-pass transmembrane notch receptor with a notch ligand in the adjacent cell (trans activation). Different ligands can activate notch, and based on their combination, their fate differs. Recent studies report that DLL1 and DLL4 notch ligands can bind the same notch receptor, in which binding of the DLL1 ligand promotes myogenesis, whereas DLL4 binding inhibits it (9,22). Intriguingly, when both the receptor and ligand are expressed in the same cell, it results in cis-inhibitory action, which limits the activity of notch (25). Ligand binding to the notch receptor triggers a series of proteolytic cleavage called regulated intramembrane proteolysis (RIP), due to the exposure of the S2 site in the heterodimerization domain. This exposure is initiated as a result of mechanical pull generated by subsequent initiation of ligand endocytosis via ligand binding or conformational change in the negative regulatory region (NRR). The exposed S2 site is then cleaved by a-disintegrin-and metalloproteinases (ADAM)-type metalloprotease. Further, the membrane-tethered truncated fragment is sensitive to subsequent cleavage by γ-secretase at the S3 site of the receptor, resulting in the release of the intracellular domain of the notch receptor from the membrane into the cytoplasm. The splitting of notch intracellular domain (NICD) from the membrane allows it to translocate to the nucleus and to assemble into a transcriptional activation complex. The main components of this complex include a DNA binding protein named CBF-1, Suppressor of Hairless, Lag-1 proteins family (CSL) (CBF1/RBP-J in mammals), Suppressor of hairless (in D. melanogaster), Lag1 (in C. elegans), NICD and a coactivator-Mastermind-like/Lag-3 family protein (19,20,26–30). In the transcriptional activation complex, CSL binds the DNA by recognizing the consensus sequence (C/T) GTGGGAA in the proximal promoter region of the notch target genes (31). The best characterized immediate downstream effectors/target genes of notch signaling are the basic helix-loop-helix (bHLH) group of transcriptional repressor—hes family bHLH transcription factor (HES) and HES-related genes, such as hairy/enhancer of split related with YRPW motif (HEY) (27,32–35). Upon activation of notch signaling, there is a competition between co-repressor molecules and NICD in cells to form the NICD-CSL complex, which flips the cell from a transcriptional repressor stage to a transcriptionally active form by recruiting various coactivators like p300, which connect the general transcriptional machinery to core notch signaling (Figure 12.2). The turnover of notch signaling is highly modulated by several modulator molecules and cross talk between various signaling pathway components. Ligand responsiveness—epsin-dependent endocytosis of ligands and the stability and half-life of NICD—depends mainly on modifications like glycosylation, hydroxylation, ubiquitylation of receptor ectodomain (ECD), phosphorylation, acetylation and deacetylation of NICD (36).
Small GTPases in platelet membrane trafficking
Published in Platelets, 2019
Tony G. Walsh, Yong Li, Andreas Wersäll, Alastair W. Poole
Of particular relevance to this review are the Ras-like (Ral) GTPases, RalA and RalB. Like most Ras GTPases, Rals have been implicated in numerous cellular processes including oncogenic transformations, but have also been implicated as regulators of vesicle trafficking [80]. They are ubiquitously expressed, with high abundance in brain, testes and platelets sharing 82% sequence identity, with ~ 55% identity to other Ras GTPases [81]. A well-characterised effector of Rals is the exocyst complex, comprising of 8 proteins (EXOC1-8) that facilitate the tethering of exocytic vesicles to the plasma membrane [82]. Notably, all components of this octameric complex are present in human (and mouse) platelets and GTP-loaded Rals have been shown to bind EXOC2 and EXOC8 in a mutually exclusive manner [83]. Similarly, other established effectors of Rals include Ral binding protein (RalBP1) and phospholipase D, but their relevance to platelet function and/or vesicle trafficking remains to be determined. Of relevance, RalBP1 has been shown to play a role in receptor-mediated endocytosis by interacting with epsin homology domain proteins Reps1 and Reps2, aswell as clathrin adaptor protein complex AP2, all of which are present in platelets and are supportive of an endocytic function for platelet Rals (Figure 1) [84].
Targeting the intestinal lymphatic system: a versatile path for enhanced oral bioavailability of drugs
Published in Expert Opinion on Drug Delivery, 2018
Renuka Suresh Managuli, Sushil Yadaorao Raut, Meka Sreenivasa Reddy, Srinivas Mutalik
Clathrin-mediated endocytosis involves the submerging of molecule into the cell plasma membrane which buds inward to form vesicles. Here, a GTPase called dynamin plays a major role in vesicle formation with involvement in the budding and scission process of nascent vesicles from parent membrane. The molecule filled budding vesicle is covered with 180 kDa proteins called clathrins which are triskelion, consisting of three heavy chains and three light chains. Many triskelion clathrins arrange into pentagon or hexagon structure surrounding the vesicle. Clathrin does not directly bind to the plasma membrane or to vesicle, instead adaptor proteins AP2, AP180, and epsin fuses clathrins to vesicles. The clathrin coating over vesicles is then detached thereby releasing the vesicles into endosomes. Synaptic vesicle uptake in neurons is an example of clathrin-mediated endocytosis [47,48]. Caveolae are cholesterol and sphingolipid rich plasma membrane’s inward growth vesicles coated with caveolin-1 protein [49]. Clathrin and caveolae independent endocytosis do not involve the clathrin or caveolae coating over pits. Chlorpromazine and nystatin are clathrin and caveolae-mediated endocytosis inhibitors, respectively, which are used in depicting the absorption mechanism of nanoparticles [50–52]. The Soluplus®/TPGS binary mixed micelle system prepared by Hu et al. [53], showed lymphatic transport in cycloheximide rat model and suggested lipid raft/caveolae and macropinocytosis-mediated the cell uptake through P-glycoprotein (P-gp)-independent pathway.
Phage display technology for target determination of small-molecule therapeutics: an update
Published in Expert Opinion on Drug Discovery, 2020
Yoichi Takakusagi, Kaori Takakusagi, Kengo Sakaguchi, Fumio Sugawara
Pyrenocine B is a natural compound [40] that inhibits the display of endogenous MHC class II-restricted antigens in bone marrow-derived dendritic cells (BMDCs). The Epsin R molecule was identified by T7 PD screening and surface plasmon resonance (SPR) analysis [41]. Epsin R is known to play a role in endosomal trafficking by binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) [42]. Downregulation of EpsinR expression in BMDCs by shRNA resulted in a decrease in the responsiveness of CD4+ T cells. Thus, they demonstrated that Epsin R has a previously unknown role in the display of endogenous MHC class II-restricted antigen, which involves SNARE-dependent sorting in the mechanism.