The amyloid beta peptide (Aβ) is derived from the amyloid precursor protein (APP) by secretase processing. APP is also cleaved by numerous other proteases, such as the type II transmembrane serine protease matriptase, with consequences on the production of Aβ. Because the APP homolog protein amyloid-like protein 1 (APLP1) shares similarities with APP, we sought to determine if matriptase also plays a role in its processing. Here, we demonstrate that matriptase directly interacts with APLP1 and that APLP1 is cleaved in cellulo by matriptase in its E1 ectodomains at arginine 124. Replacing Arg124 with Ala abolished APLP1 processing by matriptase. Using a bioluminescence resonance energy transfer (BRET) assay we found that matriptase reduces APLP1 homodimeric interactions. This study identifies matriptase as the first protease cleaving APLP1 in its dimerization domain, potentially altering the multiple functions associated with dimer formation. Alzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive and accelerated loss of neurons, leading to cognitive disorders and is currently the most common dementia 1. Accumulation of extracellular amyloid beta (Aβ) whether in the form of plaques, oligomers or soluble monomers is a fundamental hallmark of AD 2,3. In the pathogenic amyloidogenic pathway, successive APP cleavages by βand γ-secretase results in the production of Aβ 4. Recent treatment strategies targeting elements of the amyloidogenic pathway have failed to slow the progression of symptoms. Therefore, a better understanding of the mechanisms involved in AD is needed and several teams have focused on the physiological role and biosynthesis/processing of APP family members. Amyloid-like protein 1 (APLP1) is part of the same family and is homologous to APP 5. According to the Human Protein Atlas 6,7 , APLP1 is enriched in the human brain while APP is ubiquitously expressed, consistent with data obtained in mice 8. APP and APLP1 are type I transmembrane proteins sharing conserved luminal E1 and E2 domains 5. The E1 domain, rich in cysteines, is comprised of two subdomains, a growth factor-like subdomain (GFLD) that binds heparin and that stimulates neurite growth, as well as a CuBD subdomain that binds Cu and Zn ions 9. The E2 domain forms an antiparallel dimer and binds heparin in its dimeric form 10. Finally, the C-terminal domains of these proteins contain a YENPTY motif that serves as an endocytosis signal 11. Although both proteins can be cleaved by secretases, the Aβ sequence is only found in APP 12,13. APP and APLP1 are both involved in neuronal differentiation, synaptogenesis, neurite growth, and synaptic plasticity 14-16. APP and APLP1 are known to form homo-and heterodimers 17 , which are in part dependent on the conserved E1 domain 18. These dimeric interactions occur at the plasma membrane on a single cell (cis interaction) but also occur between transmembrane proteins of adjacent cells (trans interaction) 19-21. APP/APLP1 interactions promote cell adhesion in a homo-and heter...
Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt–villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.
The complex and dynamic interplay between internalization, anterograde transport, recycling and degradation determines the density of functional G protein-coupled receptors (GPCRs) at the cell surface and, consequently, the magnitude of their associated physiological responses. As opposed to most members of the GPCR superfamily, the delta opioid receptor (DOP) is only weakly expressed at the neuronal plasma membrane, thus representing a critical limitation for its use as a therapeutic target. Although DOP appears as a promising candidate for the development of better-tolerated analgesics, the molecular and cellular mechanisms underlying the regulation of its cell surface expression remain poorly characterized. This work investigates the constitutive ( i.e. ligand-independent) trafficking of DOP, an understudied cellular process potentially involved in the control of plasma membrane-localized receptors. In HEK293 cells stably expressing Flag-tagged DOP, we first confirmed that this GPCR is constitutively internalized through a clathrin-dependent and b-arrestin-independent mechanism. Immunofluorescence experiments with selected Rab protein isoforms indicated that internalized DOP was mainly colocalized with the early endosome marker Rab5, as well as the rapid recycling endosome marker Rab4. Co-transfection with Rab5 dominant-negative mutant inhibited the intracellular distribution of the receptor, indicating that its constitutive endocytosis is Rab5-dependent. DOP cell surface expression and ligand-induced signaling were also significantly reduced following Rab4-specific DsiRNA treatments, suggesting a role for this small GTPase in the regulation of DOP constitutive recycling. Mapping of the major region of interaction between DOP and both Rabs revealed that Rab4 binds the third intracellular loop of DOP, whereas Rab5 seems to preferentially interact with the distal region of the C-terminal end of DOP. Altogether, these results show for the first time that DOP constitutive internalization and recycling are critical to maintain its cell surface bioavailability and responsiveness to agonists.
To investigate the structure-cellular penetration relationship of guanidinium-rich transporters (GRTs), we previously designed PGua4, a five-amino acid peptoid containing a conformationally restricted pattern of eight guanidines, which showed high cell-penetrating abilities and low cell toxicity. Herein, we characterized the cellular uptake selectivity, internalization pathway, and intracellular distribution of PGua4, as well as its capacity to deliver cargo. PGua4 exhibits higher penetration efficiency in HeLa cells than in six other cell lines (A549, Caco-2, fibroblast, HEK293, Mia-PaCa2, and MCF7) and is mainly internalized by clathrin-mediated endocytosis and macropinocytosis. Confocal microscopy showed that it remained trapped in endosomes at low concentrations but induced pH-dependent endosomal membrane destabilization at concentrations ≥10 μM, allowing its diffusion into the cytoplasm. Importantly, PGua4 significantly enhanced macropinocytosis and the cellular uptake and cytosolic delivery of large IgGs following noncovalent complexation. Therefore, in addition to its peptoid nature conferring high resistance to proteolysis, PGua4 presents characteristics of a promising tool for IgG delivery and therapeutic applications.
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