In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin). TOR inhibition elicits autophagy, the ...large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.
Background & Aims: Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are ...differentially expressed consistent with a potential role in creating these variable properties. Methods: Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine. Results: All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis. Conclusions: Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.
GASTROENTEROLOGY 2001;120:411-422
Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their ...molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.
Highlights • Progranulin is a secreted glycoprotein found in plasma and CSF. • Progranulin has growth factor-like and inflammation-related properties. • Progranulin deficiency causes a ...neurodegenerative disease – frontotemporal dementia. • Obese and type 2 diabetic individuals have increased serum progranulin levels. • Grn −/− mice are protected from diet-induced obesity and insulin resistance.
SUMOylation of claudin-2 Van Itallie, Christina M.; Mitic, Laura L.; Anderson, James M.
Annals of the New York Academy of Sciences,
July 2012, Volume:
1258, Issue:
1
Journal Article
Peer reviewed
Open access
The C‐terminal cytoplasmic tails of claudins are likely sites for interaction with proteins that regulate their function. We performed a yeast two‐hybrid screen with the tail of human claudin‐2 ...against a human kidney cDNA library and identified interactions with the PDZ3 domain of ZO‐2 as well as ubiquitin‐conjugating enzyme E2I (SUMO ligase‐1) and E3 SUMO‐protein ligase PIAS; the first is a predicted interaction, while the latter two are novel and suggest that claudin‐2 is a substrate for SUMOylation. Using an in vitro SUMOylation assay, we identified K218 as a conjugation site on claudin‐2; mutation of that lysine to arginine blocked SUMOylation. Stable expression of inducible GFP‐SUMO‐1 in MDCK cells resulted in decreased levels of claudin‐2 protein by immunoblot and decreased claudin‐2 membrane expression by immunofluorescence microscopy. We conclude that the cellular levels of claudin‐2 may be modulated by SUMOylation, warranting further investigation of cellular pathways that regulate this modification in vivo.
Tight junctions form the major paracellular barrier in epithelial tissues. Barrier-sealing properties are quite variable among cell types in terms of electrical resistance, solute and water flux, and ...charge selectivity. A molecular explanation for this variability appears closer following identification of the transmembrane proteins occludin and members of the claudin multigene family. For example, the human phenotype of mutations in claudin-16 suggests that it creates a channel that allows magnesium to diffuse through renal tight junctions. Similarly, a mouse knockout of claudin-11 reveals its role in formation of tight junctions in myelin and between Sertoli cells in testis. The study of other claudins is expected to elucidate their contributions to creating junction structure and physiology in all epithelial tissues.
Three types of transmembrane proteins have been identified within the tight junction, but it remains to be determined how they provide the molecular basis for regulating the paracellular permeability ...for water, solutes, and immune cells. Several of these proteins localize specifically within the continuous cell-to-cell contacts of the tight junction. One of these, occludin, is a cell adhesion molecule that has been demonstrated to influence ion and solute permeability. The claudins are a family of four-membrane spanning proteins; unexpectedly, other members of this family have already been characterized without recognizing their relationship to tight junctions. Junction adhesion molecule, the most recently identified tight junction component, is a member of the Ig superfamily and influences the paracellular transmigration of immune cells. A plaque of cytoplasmic proteins under the junction may be responsible for scaffolding the transmembrane proteins, creating a link to the perijunctional actin cytoskeleton and transducing regulatory signals that control the paracellular barrier.
The tight junction tetraspan protein claudin‐4 creates a charge‐selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from ...transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin‐4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro‐octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin‐4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique.
Frontotemporal lobar degeneration–causing mutations in the progranulin (GRN) gene reduce progranulin protein (PGRN) levels, suggesting that restoring PGRN in mutation carriers may be therapeutic. ...Nimodipine, a Food and Drug Administration–approved blood-brain barrier-penetrant calcium channel blocker, increased PGRN levels in PGRN-deficient murine models. We sought to assess safety and tolerability of oral nimodipine in human GRN mutation carriers.
We performed an open-label, 8-week, dose-finding, phase 1 clinical trial in eight GRN mutation carriers to assess the safety and tolerability of nimodipine and assayed fluid and radiologic markers to investigate therapeutic endpoints.
There were no serious adverse events; however, PGRN concentrations (cerebrospinal fluid and plasma) did not change significantly following treatment (percent changes of −5.2 ± 10.9% in plasma and −10.2 ± 7.8% in cerebrospinal fluid). Measurable atrophy within the left middle frontal gyrus was observed over an 8-week period.
While well tolerated, nimodipine treatment did not alter PGRN concentrations or secondary outcomes.
Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier ...requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.
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