Lipid regulation of Atg8 family protein recruitment and membrane modulation of mitophagy and autophagic cell death

299 p. (eng.) 307 p.(eusk.)Tesi lan honen aurkikuntzek, mitofagian zehar LC3/GABARAP familiako proteinen mitokondrioetarako errekrutatze-prozesuaren mekanismo molekularraren eta Atg8 proteinaren giza ortologoek giza lerro zelular ezberdinetan daukaten funtzioaren inguruko argibideak ematen dituzte. Kardiolipinak, kaltetutako mitokondrioak hautatzeko errezeptore gisa daukan funtzioa, zenbait faktoreren ondorioz erregulatzen da. Horien artean aurkitzen dira indar ionikoa, bigeruzaren jariakortasuna eta mintzaren kurbadura. Proteina-lipido elkarrekintza elektrostatiko hau, mitofagiarekiko espezifikoa izango litzateke eta ez litzateke autofagia babesgarri ez-hautakorrean edo THC kanabinoideak eragindako heriotza zelular autofagikoan gertatuko. Gainera, LC3/GABARAP proteina familiako kide bakoitzak, zelula-motaren menpekoa den mekanismoaren bidez (estresarekiko espezifikoa ere izan litekeena), mitokondriora erakarriak dira (Antón eta lank., 2016; Antón eta lank., prestatzen). Azkenik, tesi lan honetan deskribatutako aurkikuntzek, THCk eragindako autofagia bidezko minbizi zelulen heriotza, erretikulu endoplasmatikoan pilatutako dihidrozeramidaren ondoriozko autolisosometako mintzaren iragazkortasunaren handipenean eta heriotza zelular apoptotikoaren aktibazioan oinarritzen dela defendatzen dute (Hernández-Tiedra eta lank., 2016). Gainera, LC3B proteinaren lipidazioak, proteinak mitokondrioaren mintzean zeramidara lotzeko daukan gaitasuna modulatzen du (Antón eta lank., 2016), horrela mitofagia bidezko heriotza zelularra eta tumoreen ezabaketa erregulatuz.Instituto de Biofisika Institutua (CSIC, UPV/EHU

Lipid regulation of Atg8 family protein recruitment and membrane modulation of mitophagy and autophagic cell death

299 p. (eng.) 307 p.(eusk.)Tesi lan honen aurkikuntzek, mitofagian zehar LC3/GABARAP familiako proteinen mitokondrioetarako errekrutatze-prozesuaren mekanismo molekularraren eta Atg8 proteinaren giza ortologoek giza lerro zelular ezberdinetan daukaten funtzioaren inguruko argibideak ematen dituzte. Kardiolipinak, kaltetutako mitokondrioak hautatzeko errezeptore gisa daukan funtzioa, zenbait faktoreren ondorioz erregulatzen da. Horien artean aurkitzen dira indar ionikoa, bigeruzaren jariakortasuna eta mintzaren kurbadura. Proteina-lipido elkarrekintza elektrostatiko hau, mitofagiarekiko espezifikoa izango litzateke eta ez litzateke autofagia babesgarri ez-hautakorrean edo THC kanabinoideak eragindako heriotza zelular autofagikoan gertatuko. Gainera, LC3/GABARAP proteina familiako kide bakoitzak, zelula-motaren menpekoa den mekanismoaren bidez (estresarekiko espezifikoa ere izan litekeena), mitokondriora erakarriak dira (Antón eta lank., 2016; Antón eta lank., prestatzen). Azkenik, tesi lan honetan deskribatutako aurkikuntzek, THCk eragindako autofagia bidezko minbizi zelulen heriotza, erretikulu endoplasmatikoan pilatutako dihidrozeramidaren ondoriozko autolisosometako mintzaren iragazkortasunaren handipenean eta heriotza zelular apoptotikoaren aktibazioan oinarritzen dela defendatzen dute (Hernández-Tiedra eta lank., 2016). Gainera, LC3B proteinaren lipidazioak, proteinak mitokondrioaren mintzean zeramidara lotzeko daukan gaitasuna modulatzen du (Antón eta lank., 2016), horrela mitofagia bidezko heriotza zelularra eta tumoreen ezabaketa erregulatuz.Instituto de Biofisika Institutua (CSIC, UPV/EHU

Human ATG3 binding to lipid bilayers: role of lipid geometry, and electric charge

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic a-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.This article is part of COST (European Cooperation in Science and Technology) Actions (PROTEOSTASIS, BM1307, TRANSAUTOPHAGY, CA15138). The authors thank Dr. Isei Tanida (National Institute of Infectious Diseases, Tokyo, Japan) for providing human ATG3 and GABARAP plasmids, and to Dr. Martin B. Ulmschneider (Johns Hopkins University, Baltimore, MD) for Fig. 1B. They are also indebted to Ms Araceli Marcos for technical support. This work was supported in part by grants from the Spanish Ministry of Economy and FEDER (BFU 2011-28566, BFU 2015-66306-P, AGL2011-24758), and from the Basque Government (IT838-13, IT84913). A.S. acknowledges support from RyC Program of the Spanish Ministry of Economy. J.H.H and Z.A. were predoctoral students supported by the University of the Basque Country. Editoria

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1\u2013dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1\u2013mediated organelle transport

ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brai

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Molecular and mesoscopic geometries in autophagosome generation. A review

Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of 'intrinsic curvature', and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.This work was supported in part by grants from the Spanish Ministry of Economy (grant FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grant No. IT1270-19), as well as by Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. MNI was supported by a FPU predoctoral contract from MINECO.Peer reviewe

Prokaryotic Capability to Use Organic Substrates Across the Global Tropical and Subtropical Ocean

15 pages, 8 figures, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2020.00918/full#supplementary-materialProkaryotes play a fundamental role in decomposing organic matter in the ocean, but little is known about how microbial metabolic capabilities vary at the global ocean scale and what are the drivers causing this variation. We aimed at obtaining the first global exploration of the functional capabilities of prokaryotes in the ocean, with emphasis on the under-sampled meso- and bathypelagic layers. We explored the potential utilization of 95 carbon sources with Biolog GN2 platesR in 441 prokaryotic communities sampled from surface to bathypelagic waters (down to 4,000 m) at 111 stations distributed across the tropical and subtropical Atlantic, Indian, and Pacific oceans. The resulting metabolic profiles were compared with biological and physico-chemical properties such as fluorescent dissolved organic matter (DOM) or temperature. The relative use of the individual substrates was remarkably consistent across oceanic regions and layers, and only the Equatorial Pacific Ocean showed a different metabolic structure. When grouping substrates by categories, we observed some vertical variations, such as an increased relative utilization of polymers in bathypelagic layers or a higher relative use of P-compounds or amino acids in the surface ocean. The increased relative use of polymers with depth, together with the increases in humic DOM, suggest that deep ocean communities have the capability to process complex DOM. Overall, the main identified driver of the metabolic structure of ocean prokaryotic communities was temperature. Our results represent the first global depiction of the potential use of a variety of carbon sources by prokaryotic communities across the tropical and the subtropical ocean and show that acetic acid clearly emerges as one of the most widely potentially used carbon sources in the oceanThis research was funded by the Spanish Ministry of Economy and Competitiveness under projects Malaspina-2010 Circumnavigation Expedition (grant number CSD2008-00077) and partly by projects ANIMA (CTM2015-65720-R), and MIAU (RTI2018-101025-B-I00). CR-G was supported by a Juan de la Cierva fellowship and the GRAMMI project (IJCI-2015-23505 and RTI2018-099740-J-I00, MICINN, Spain)With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI

Prokaryotic capability to use organic substrates across the ocean

Trabajo presentado al ASLO Aquatic Sciences Meeting, celebrado virtualmente del 22 al 27 de junio de 2021.Peer reviewe