Autophagy and endocytosis - interconnections and interdependencies.

Autophagy and endocytosis are membrane-vesicle-based cellular pathways for degradation and recycling of intracellular and extracellular components, respectively. These pathways have a common endpoint at the lysosome, where their cargo is degraded. In addition, the two pathways intersect at different stages during vesicle formation, fusion and trafficking, and share parts of the molecular machinery. Accumulating evidence shows that autophagy is dependent upon endocytosis and vice versa. The emerging joint network of autophagy and endocytosis is of vital importance for cellular metabolism and signaling, and thus also highly relevant in disease settings. In this Review, we will discuss examples of how the autophagy machinery impacts on endocytosis and cell signaling, and highlight how endocytosis regulates the different steps in autophagy in mammalian cells. Finally, we will focus on the interplay of these pathways in the quality control of their common endpoint, the lysosome.

FKBP8 recruits LC3A to mediate Parkin-independent mitophagy.

Mitophagy, the selective removal of damaged or excess mitochondria by autophagy, is an important process in cellular homeostasis. The outer mitochondrial membrane (OMM) proteins NIX, BNIP3, FUNDC1, and Bcl2-L13 recruit ATG8 proteins (LC3/GABARAP) to mitochondria during mitophagy. FKBP8 (also known as FKBP38), a unique member of the FK506-binding protein (FKBP) family, is similarly anchored in the OMM and acts as a multifunctional adaptor with anti-apoptotic activity. In a yeast two-hybrid screen, we identified FKBP8 as an ATG8-interacting protein. Here, we map an N-terminal LC3-interacting region (LIR) motif in FKBP8 that binds strongly to LC3A both in vitro and in vivo FKBP8 efficiently recruits lipidated LC3A to damaged mitochondria in a LIR-dependent manner. The mitophagy receptors BNIP3 and NIX in contrast are unable to mediate an efficient recruitment of LC3A even after mitochondrial damage. Co-expression of FKBP8 with LC3A profoundly induces Parkin-independent mitophagy. Strikingly, even when acting as a mitophagy receptor, FKBP8 avoids degradation by escaping from mitochondria. In summary, this study identifies novel roles for FKBP8 and LC3A, which act together to induce mitophagy.

Fluorescence microscopy and correlative brightfield videos of mitochondria and vesicles in H9c2 cardiomyoblasts.

This paper presents data acquired to study the dynamics and interactions of mitochondria and subcellular vesicles in living cardiomyoblasts. The study was motivated by the importance of mitochondrial quality control and turnover in cardiovascular health. Although fluorescence microscopy is an invaluable tool, it presents several limitations. Correlative fluorescence and brightfield images (label-free) were therefore acquired with the purpose of achieving virtual labelling via machine learning. In comparison with the fluorescence images of mitochondria, the brightfield images show vesicles and subcellular components, providing additional insights about sub-cellular components. A large part of the data contains correlative fluorescence images of lysosomes and/or endosomes over a duration of up to 400 timepoints (>30 min). The data can be reused for biological inferences about mitochondrial and vesicular morphology, dynamics, and interactions. Furthermore, virtual labelling of mitochondria or subcellular vesicles can be achieved using these datasets. Finally, the data can inspire new imaging experiments for cellular investigations or computational developments. The data is available through two large, open datasets on DataverseNO.

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA.

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.

Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA.

The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

The NRF2 activator DH404 attenuates adverse ventricular remodeling post-myocardial infarction by modifying redox signalling.

BACKGROUND: The novel synthetic triterpenoid, bardoxolone methyl, has the ability to upregulate cytoprotective proteins via induction of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway. This makes it a promising therapeutic agent in disease states characterized by dysregulated oxidative signalling. We have examined the effect of a Nrf2 activator, dihydro-CDDO-trifluoroethyl amide (DH404), a derivative of bardoxolone methyl, on post-infarct cardiac remodeling in rats. METHODS/RESULTS: DH404, administered from day 2 post myocardial infarction (MI: 30min transient ischemia followed by reperfusion) resulted in almost complete protection against adverse ventricular remodeling as assessed at day 28 (left ventricular end-systolic area: sham 0.14±0.01cm2, MI vehicle 0.29±0.04cm2 vs. MI DH404 0.18±0.02cm2, P<0.05); infarct size (21.3±3.4% MI vehicle vs. 10.9±2.3% MI DH404, P<0.05) with associated benefits on systolic function (fractional shortening: sham 71.9±2.6%, MI vehicle 36.2±1.9% vs. MI DH404 58.6±4.0%, P<0.05). These structural and functional benefits were associated with lower myocardial expression of atrial natriuretic peptide (ANP, P<0.01 vs. MI vehicle), and decreased fibronectin (P<0.01 vs. MI vehicle) in DH404-treated MI rats at 28 days. MI increased glutathionylation of endothelial nitric oxide synthase (eNOS) in vitro - a molecular switch that uncouples the enzyme, increasing superoxide production and decreasing nitric oxide (NO) bioavailability. MI-induced eNOS glutathionylation was substantially ameliorated by DH404. An associated increase in glutaredoxin 1 (Grx1) co-immunoprecipitation with eNOS without a change in expression was mechanistically intriguing. Indeed, in parallel in vitro experiments, silencing of Grx1 abolished the protective effect of DH404 against Angiotensin II-induced eNOS uncoupling. CONCLUSION: The bardoxolone derivative DH404 significantly attenuated cardiac remodeling post MI, at least in part, by re-coupling of eNOS and increasing the functional interaction of Grx1 with eNOS. This agent may have clinical benefits protecting against post MI cardiomyopathy.

RNA reprogramming and repair based on trans-splicing group I ribozymes.

While many traditional gene therapy strategies attempt to deliver new copies of wild-type genes back to cells harboring the defective genes, RNA-directed strategies offer a range of novel therapeutic applications. Revision or reprogramming of mRNA is a form of gene therapy that modifies mRNA without directly changing the transcriptional regulation or the genomic gene sequence. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence-specific manner, and to covalently link a new RNA sequence to this separate RNA molecule. Group I ribozymes have been shown to repair defective transcripts that cause human genetic or malignant diseases, as well as to replace transcript sequences by foreign RNA resulting in new cellular functions. This review provides an overview of current strategies using trans-splicing group I ribozymes in RNA repair and reprogramming.

Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing.

DiGIR2 is the group I splicing-ribozyme of the mobile twin-ribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3'-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3'-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.