The autophagy machinery interacts with EBV capsids during viral envelope release.

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

Extracellular vesicles (EVs) are secreted by all living cells and are found in body fluids. They exert numerous physiological and pathological functions and serve as cargo shuttles. Due to their safety and inherent bioactivity, they have emerged as versatile therapeutic agents, biomarkers, and potential drug carriers. Despite the growing interest in EVs, current progress in this field is, in part, limited by relatively inefficient isolation techniques. Conventional methods are indeed slow, laborious, require specialized laboratory equipment, and may result in low yield and purity. This work describes an electrochemically controlled "all-in-one" device enabling capturing, loading, and releasing of EVs. The device is composed of a fluidic channel confined within antibody-coated microstructured electrodes. It rapidly isolates EVs with a high level of purity from various biofluids. As a proof of principle, the device is applied to isolate EVs from skin wounds of healthy and diabetic mice. Strikingly, it is found that EVs from healing wounds of diabetic mice are enriched in mitochondrial proteins compared to those of healthy mice. Additionally, the device improves the loading protocol of EVs with polyplexes, and may therefore find applications in nucleic acid delivery. Overall, the electrochemical device can greatly facilitate the development of EVs-based technologies.

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0-4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy.

The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.

Comparison of anthropometric, cardiovascular, autonomic, baroreflex sensitivity, aerobic fitness, inflammatory markers and oxidative stress parameters between first degree relatives of diabetes and controls.

AIM: We aimed at assessing cardiovascular risk of first degree relatives of diabetes (FDRD). METHODS: A cross sectional study involving 90 apparently healthy normoglycemic volunteers aged between 15 and 50 years (45 FDRD and 45 FDRs of non-diabetics). We measured anthropometric parameters, baroreflex sensitivity, heart rate variability, cardiac autonomic function tests, and aerobic capacity, fasting blood glucose and insulin, lipid profile, inflammatory markers, nitric oxide and oxidative stress markers. RESULTS: FDRD had significantly higher hip circumference and BF%. Blood pressure, total peripheral resistance and cardiac output were comparable. FDRD had higher HR and rate pressure product. There were no significant differences in cardio-respiratory fitness (VO2max) and physical activity level. Time and Frequency domain parameters were comparable except for reduced NN50 and total power. Baroreflex sensitivity, 30:15 ratio and E: I ratio were significantly less in FDRD. Fasting glucose was comparable. Fasting Insulin, HOMA IR, HOMA %B and HOMA AD were higher while HOMA %S and QUICKI index were lower in FDRD. Lipid profile or lipid derived parameters were comparable except for higher non-HDLc in FDRD. Adiponectin was lower while Leptin and Leptin/apidonectin ratio was higher in FDRD. IL-6, hsCRP, TNF- alpha and MDA were significantly higher in FDRD, while TAS and nitric oxide were significantly lower in FDRD. CONCLUSION: Higher body fat percentage, with insulin resistance, deranged cardiac autonomic function, higher oxidative stress and inflammation, lower adiponectin and nitric oxide levels places FDRD at higher cardiovascular risk and necessitates early lifestyle modification/intervention.