Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, whereO-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.

Rationale: Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/β-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored. Objectives: To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD. Methods: FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase–polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/β-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components. Measurements and Main Results: FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin–driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/β-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo. Conclusions: Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

The pathological conversion of cellular prion protein (PrP^C) into the scrapie prion protein (PrP^Sc) isoform appears to have a central role in the pathogenesis of transmissible spongiform encephalopathies. However, the identity of the intracellular compartment where this conversion occurs is unknown. Several lines of evidence indicate that detergent-resistant membrane domains (DRMs or rafts) could be involved in this process. We have characterized the association of PrP^C to rafts during its biosynthesis. We found that PrP^C associates with rafts already as an immature precursor in the endoplasmic reticulum. Interestingly, compared with the mature protein, the immature diglycosylated form has a different susceptibility to cholesterol depletion vs. sphingolipid depletion, suggesting that the two forms associate with different lipid domains. We also found that cholesterol depletion, which affects raft-association of the immature protein, slows down protein maturation and leads to protein misfolding. On the contrary, sphingolipid depletion does not have any effect on the kinetics of protein maturation or on the conformation of the protein. These data indicate that the early association of PrP^C with cholesterol-enriched rafts facilitates its correct folding and reinforce the hypothesis that cholesterol and sphingolipids have different roles in PrP metabolism.

Short peptides or single amino acids are interesting building blocks for fabrication of hydrogels, frequently used as extracellular matrix-mimicking scaffolds for cell growth in tissue engineering. The combination of two or more peptide hydrogelators could allow obtaining different materials exhibiting new architectures, tunable mechanical properties, high stability and improved biofunctionality. Here we report on the synthesis, formulation and multi-scale characterization of peptide-based mixed hydrogels formed by the low molecular weight Fmoc-FF (N^α-fluorenylmethyloxycarbonyl diphenylalanine) hydrogelator and of the PEG₈-(FY)3 hexapeptide, containing three repetitions of the Phe-Tyr motif and a PEG moiety at its N-terminus. Mixed hydrogels were also prepared by replacing PEG₈-(FY)3 with its analogue (FY)3, without the PEG moiety. Rheology analysis confirmed the improved mechanical features of the multicomponent gels prepared at two different ratios (2/1 or 1/1, v/v). However, the presence of the hydrophilic PEG polymeric moiety causes a slowing down of the gel kinetic formation (from 42 to 18 minutes) and a decrease of the gel rigidity (G′ from 9 to 6 kPa). Preliminary in vitro biocompatibility and cell adhesion assays performed on Chinese hamster ovarian (CHO) cells suggest a potential employment of these multicomponent hydrogels as exogenous scaffold materials for tissue engineering.

The coronavirus infection is constantly diffusing worldwide and the incidence of death is dramatically increasing, representing one of the greatest disasters in human history. Nowadays, no effective therapeutic approaches have been licensed, despite the rising interest of the scientific research in this specific field, and the daily growing number of publications, while the need to find novel strategies is urgent. Evidence in the literature reported the antiviral activity of polyphenols, the largest class of bioactive compounds in nature. Interestingly, a limited number of studies investigated the efficacy of polyphenols from different raw materials, directly against coronaviruses. The present manuscript aimed to report this evidence and provide a viewpoint on the possibility to use it as a start point for the development of novel natural approaches against this viral infection, eventually designing further appropriate researches.