Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, ...identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTPCI73T). Comparing syngeneic mutant versus gene-corrected iPSCs after differentiation into AEC2s (iAEC2s), we find that mutant iAEC2s accumulate large amounts of misprocessed and mistrafficked pro-SFTPC protein, similar to in vivo changes, resulting in diminished AEC2 progenitor capacity, perturbed proteostasis, altered bioenergetic programs, time-dependent metabolic reprogramming, and nuclear factor κB (NF-κB) pathway activation. Treatment of SFTPCI73T-expressing iAEC2s with hydroxychloroquine, a medication used in pediatric ILD, aggravates the observed perturbations. Thus, iAEC2s provide a patient-specific preclinical platform for modeling the epithelial-intrinsic dysfunction at ILD inception.
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•Patient-specific iAEC2s serve as a human preclinical platform for modeling ILD•Patient-derived iAEC2s expressing mutant SFTPCI73T have diminished progenitor capacity•SFTPCI73T mutant iAEC2s display proteostasis perturbations and metabolic reprogramming•Mutant iAEC2s are an important proinflammatory hub via activation of the NF-κB pathway
Alysandratos et al. differentiate patient-specific iPSCs carrying the ILD-associated SFTPCI73T variant and syngeneic corrected iPSCs into alveolar epithelial type 2 cells (iAEC2s). They find that mutant iAEC2s demonstrate proteostasis perturbations, altered bioenergetic programs, and metabolic reprogramming. They validate these findings in vivo in primary mouse SftpcI73T AEC2s.
Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the ...striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.
Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but ...are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.
An interaction between arrestin-3 and MKK7 has never been elucidated.
Results: Arrestin-3 directly binds MKK7 and promotes JNK3α2 phosphorylation by MKK7 in vitro and in intact cells.
Conclusion: ...Arrestin-3 recruits JNK3α2 and both upstream MKKs.
Significance: Arrestin-3 promotes full JNK3α2 activation; MKK binding is regulated by JNK3α2.
Arrestin-3 was previously shown to bind JNK3α2, MKK4, and ASK1. However, full JNK3α2 activation requires phosphorylation by both MKK4 and MKK7. Using purified proteins we show that arrestin-3 directly interacts with MKK7 and promotes JNK3α2 phosphorylation by both MKK4 and MKK7 in vitro as well as in intact cells. The binding of JNK3α2 promotes an arrestin-3 interaction with MKK4 while reducing its binding to MKK7. Interestingly, the arrestin-3 concentration optimal for scaffolding the MKK7-JNK3α2 module is ∼10-fold higher than for the MKK4-JNK3α2 module. The data provide a mechanistic basis for arrestin-3-dependent activation of JNK3α2. The opposite effects of JNK3α2 on arrestin-3 interactions with MKK4 and MKK7 is the first demonstration that the kinase components in mammalian MAPK cascades regulate each other's interactions with a scaffold protein. The results show how signaling outcomes can be affected by the relative expression of scaffolding proteins and components of signaling cascades that they assemble.
Biological Role of Arrestin-1 Oligomerization Samaranayake, Srimal; Vishnivetskiy, Sergey A; Shores, Camilla R ...
The Journal of neuroscience,
10/2020, Letnik:
40, Številka:
42
Journal Article
Recenzirano
Odprti dostop
Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 ...oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.
Visual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating that
monomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer.
Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem ...cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
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•Reporter lines allow detailed profiling of pluripotent stem cell-derived airway•PSC-derived airway spheres contain basal and secretory cells as well as non-lung•Single-cell RNA-seq reveals heterogeneity and potential plasticity
In this article, Kotton and colleagues show that human pluripotent stem cell-derived airway epithelial spheres contain basal and secretory airway cells. Using reporter lines for the secretory airway lineage alongside transcriptomic and functional analyses, they demonstrate that these cells have characteristic features of this population but are susceptible to Wnt-dependent phenotypic drift toward an alveolar type II cell-like program.
Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins ...have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting ...receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRETmax values for the M2 muscarinic acetylcholine (M2R), β2-adrenergic (β2AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β2AR mutants bound arrestins at 20–50% of the level of wild type β2AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β2AR than M2R and D2 dopamine receptor.
The relative contribution of phosphates and active GPCR conformation is unknown.
Using WT and mutant arrestins and receptors, we show that phosphates are critical for arrestin binding to some GPCRs but not to others.
The role of receptor-attached phosphates in arrestin binding varies widely depending on the arrestin-receptor combination.
Distinct molecular mechanisms mediate arrestin recruitment to different GPCRs.
Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for ...FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin. Cells lacking both nonvisual arrestins showed excessive spreading on fibronectin and poly-d-lysine, increased adhesion, and reduced motility. The absence of arrestins greatly increases the size and lifespan of FAs, indicating that arrestins are necessary for rapid FA turnover. In nocodazole washout assays, FAs in arrestin-deficient cells were unresponsive to disassociation or regrowth of microtubules, suggesting that arrestins are necessary for microtubule targeting-dependent FA disassembly. Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells. In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype. Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.
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