Endocytosed proteins

Chatterjee, S., Smith, E. R., Hanada, K., Stevens, V. L., and Mayor, S. (2001). GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment. EMBO J. 20(7), 1583-1592. 

Active transport mechanisms exist in the gastrointestinal tract and other epithelial sites, for the absorption of di- and tri-peptides. As described above, a greater understanding of the molecular specificity of this carrier could provide important leads for the delivery of peptides. Proteins and large peptides may be transported across cells via endocytic processes. Transcytosis is achieved if the endocytic vesicles can reach the basal membrane without fusion with lysosomes. However, various studies have shown that in the majority of cases the internalized protein is degraded, indicating that the transcytotic pathway is a minor one and most of the endocytosed protein is subject to lysosomal degradation. 

Inherited defects in amino acid transport affect epithelial cells of the gastrointestinal tract and renal tubules. Some affect transport of neutral amino acids Hartnup disease), others that of basic amino acids and ornithine and cystine (cystinuria), or of glycine and proline (Chapter 12). Cystinosis is an intracellular transport defect characterized by high intralysosomal content of free cystine in the reticuloendothelial system, bone marrow, kidney, and eye. After degradation of endocytosed protein to amino acids within lysosomes, the amino acids normally are transported to the cytosol. The defect in cystinosis may reside in the ATP-dependent efflux system for cystine transport, and particularly in the carrier protein. 

A related study by Herman (1990) with CaCo-2 cells confirmed HRP transport by endoc osis, primarily along a degradative pathway most likely associated with the lysosomal system. Other work with CaCo-2 cells and HRP (Hidalgo et al, 1989) found that HRP is endocytosed by a fluid-phase mechanism this proposed uptake mechanism was supported by the Heyman study, in which monensin was found to have no effect on HRP transcytosis. The degree of HRP degradation also varied depending on whether the marker was presented at the apical or the basal membrane of the CaCo-2 cells. Different cell types of intestinal epithelium, therefore, could process endocytosed proteins in disparate ways (Heyman et al, 1990). 

Protein toxins acting intracellularly are often composed of two subunits (A/B model). One subunit is catalytic (A-subunit) and the other is responsible for binding and cell entry (B-subunit). Following binding to an extracellular membrane receptor, the toxins are endocytosed. From the endosomes, the A-subunit is directly (pH dqDendent) transferred into the cytosol (e.g., diphtheria toxin and anthrax toxin) or the toxin is transported in a retrograde manner via the golgi to the ER (e.g., cholera toxin), where translocation into the cytosol occurs [1]. 

Cell-membrane proteins that endocytose oxidatively or otherwise modified low-density lipoproteins. 

These data suggest that one of possible mechanisms of carotenoid delivery to the neural retina may involve lipoprotein uptake from the basal side of the RPE followed by its retro-endocytosis on the apical site (Lorenzi et al., 2008). Alternatively, the endocytosed lipoprotein may be degraded in the RPE followed by secretion of certain lipophilic components from the lipoprotein at the apical site. Due to low solubility of carotenoids in aqueous solutions, it may be suggested that they are secreted already bound to a protein or that an acceptor protein is available in the interphotoreceptor matrix and/or POS. 

After initial filtration many proteins are actively reabsorbed (endocytosed) by the proximal tubules and subjected to lysosomal degradation, with subsequent amino acid reabsorption. Thus, very little intact protein actually enters the urine. 

As noted above, synaptic vesicles are not typically generated at the level of the TGN. Instead, they are assembled from endocytosed material retrieved from the synaptic plasma membrane. Synaptic vesicle and plasma membrane lipids and proteins are synthesized in the endoplasmic reticulum and modified in the Golgi apparatus, where they are then packaged in secretory vesicles. These synaptic precursors are delivered to the plasma membrane from the cell body by the constitutive secretory pathway. Synaptic vesicle proteins must be retrieved by clathrin-mediated synaptic vesicle endocytosis, a variant of RME with some neuron-specific components. Once the vesicle sheds its clathrin coat, the uncoated vesicle fuses with a... 

The problem we have not yet touched upon is how components can specifically move from one cellular component to another. Both the entry and the exit of SFV spike proteins are dependent on a number of such cellular processes. The newly synthesized spike proteins move from the ER to the Golgi complex and then to the cell surface. The cell surface membrane is continuously retrieved by endocytosis into endosomes. From here the endocytosed membrane components probably recycle back to the cell surface, but some components may also be channeled into lysosomes for degradation. Especially in cells with secretory activity, the recycling pathway from the cell surface also includes the Golgi complex (see Farquhar and Palade, 1981). 

Based on the knowledge of the endocytotic pathway, it is reasonable to assume that uhiquitination is reversible until the endocytosed membrane proteins such as neurotransmitter receptors are routed to the multivesicular body for lysosomal degradation. 

Figure 8 Ubiquitin and endocytosis. Receptors on the plasma membrane undergo monoubiquitination as a result of ligand (e.g., neurotransmitter). Ubiquitinated receptors bind to proteins called epsins, which in turn interact with adaptor proteins (adaptin) bound to clathrin-coated pits. Ubiquitination also functions to sort the internalized membrane protein into early endosomes, which directs them to degradation by lysosome through the multivesicular body. If ubiquitin from the endocytosed receptors is removed by an UBP, the receptor recycles back to the membrane. Proteasome inhibitors block endocytotic degradation of some proteins such as glutamate receptor subunits indicating a possible role for the proteasome.

The viability and function tests described above are used to evaluate the hepatocytes within the slice. Up to now, tests to measure the viability of the non-parenchymal cells have not been reported. The presence of the latter cell types is one of the conceptual advantages of slices as compared to isolated hepatocytes. As some drug targeting devices are designed to target non-parenchymal cells in the liver, the development of tests for the sinusoidal cell types deserves more attention. For example, the uptake of substrates such as succinylated human serum albumin (Suc-HSA,which is specifically endocytosed by endothelial cells [79]), or hyaluronic acid [80], can be used to assess the functionality of endocytotic pathways in the endothelial cells in the liver [81]. Other modified proteins that are specifically taken up by Kupffer cells such as mannosylated HSA, may be used to assess the functionality of the endocytotic pathway in Kupffer cells [79]. Another parameter which can be used to assess the functionality of these non-parenchymal liver cells, is the excretion of cytokines in response to pro-inflammatory stimuli. Non-parench5mal cell function in liver slices will be described in more detail in the Section 12.7. 

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