Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL’s inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL’s anti-apoptotic function.The work was supported by Grants from the Research Foundation—Flanders (FWO) (G.0901.18N), by the Research Council of the KU Leuven (OT14/101, C14/19/099, C14/19/101, and AKUL/19/34), the Interuniversity Attraction Poles Program (Belgian Science Policy; IAP-P7/13), the Central European Leuven Strategic Alliance (CELSA/18/040), and the Canadian Institutes Health Research (FDN143312). NR and HI are recipient of postdoctoral fellowships of the FWO; HI obtained a travel grant from the FWO to perform work in DIY’s laboratory. GB, JBP and DIY are part of the FWO Scientific Research Network CaSign (W0.019.17N). Work in DIY’s lab is supported by NIH (NIDCR) grant DE014756. DWA holds the Tier 1 Canada Research Chair in Membrane Biogenesis. The Switch laboratory was supported by the Flanders institute for Biotechnology (VIB), the University of Leuven, the Fund for Scientific Research Flanders (Hercules Foundation/FWO AKUL/15/34—G0H1716N). NL is funded by the Stichting Alzheimer Onderzoek (SAO-FRA 2020/0013) and is recipient of FWO postdoctoral fellowships (12P0919N and 12P0922N to NL)

Structural studies of peptide-analogues of proteins responsible for amyloidoses and for the formation of functional amyloids

Amyloid fibrils are formed by soluble proteins/peptides that convert under certain denaturing conditions into insoluble fibrous aggregates. Several proteins with important but otherwise unrelated functions have been associated with amyloid deposition, although they present neither sequence nor structural similarities. A number of widespread diseases, such as AL, AA or ATTR amyloidosis, neurodegenerative diseases (Alzheimer’s, Parkinson’s or Creutzfeldt-Jakob’s disease), type-II diabetes and several other major pathological conditions are a consequence of unrestrained deposition of amyloid causing tissue degeneration. However, organisms spanning from bacteria to humans occasionally produce functional amyloids, in an effort to support pivotal biological processes. Structural studies reveal that short sequence stretches with high aggregation propensity promote the overall amyloidogenic tendency of a protein. Such “aggregation-prone” regions, named amyloidogenic determinants, are responsible for the self-aggregation of proteins associated with the formation of amyloids. This thesis was focused towards the identification and experimental verification of “aggregation-prone” segments in amyloidogenic proteins associated with the formation of both functional and pathological amyloids. An integrative structural/biophysical approach was carried out, utilizing transmission electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy. Furthermore, computational studies were performed utilizing several bioinformatics techniques. The above were combined in an effort to uncover the unknown underlying mechanisms behind the formation and structure of amyloid fibrils.Σφαιρικές υδατοδιαλυτές πρωτεΐνες, υπό ορισμένες συνθήκες, αδυνατούν να αποκτήσουν τη φυσιολογική τους στερεοδιάταξη στο χώρο. Ως αποτέλεσμα τα πρωτεϊνικά μόρια αυτοσυγκροτούνται σε ινίδια που εμφανίζουν χαρακτηριστικές ιδιότητες και ονομάζονται αμυλοειδή. Τα αμυλοειδή ινίδια σχηματίζουν εξωκυτταρικές ή/και ενδοκυτταρικές εναποθέσεις που προκαλούν την καταστροφή κυττάρων/ιστών και σχετίζονται με μια ετερογενή ομάδα ασθενειών, τις αμυλοειδώσεις. Οι αμυλοειδώσεις περιλαμβάνουν, μεταξύ άλλων, τις νευροεκφυλιστικές νόσους του Alzheimer, του Parkinson, τις σπογγώδεις εγκεφαλοπάθειες, τον διαβήτη τύπου ΙΙ, ακόμη και ορισμένους τύπους καρκίνου. Συχνά, οργανισμοί από τα βακτήρια έως και τον άνθρωπο παράγουν φυσικά λειτουργικά αμυλοειδή, τα οποία εκμεταλλεύονται για να εξυπηρετήσουν πολύπλοκες βιολογικές διεργασίες, απαραίτητες για την ομαλή επιβίωση τους. Μικρές αλληλουχίες, γνωστές ως αμυλοειδογόνοι καθοριστές, τμήματα των αμυλοειδογόνων πρωτεϊνών, διακρίνονται για την αυξημένη ροπή που εμφανίζουν προς συσσωμάτωση. Η παρούσα διδακτορική διατριβή επικεντρώθηκε στην αναζήτηση και πειραματική επιβεβαίωση αμυλοειδογόνων καθοριστών πρωτεϊνών που σχετίζονται με τη δημιουργία τόσο λειτουργικών όσο και παθολογικών αμυλοειδών. Οι αμυλοειδογόνες ιδιότητες των αναγνωρισμένων περιοχών μελετήθηκαν και επιβεβαιώθηκαν με τη χρήση βιοφυσικών μεθόδων, όπως είναι η ηλεκτρονική μικροσκοπία διέλευσης, η περίθλαση ακτίνων-Χ, η φασματοσκοπία υπερύθρου και η πολωτική μικροσκοπία. Σε επόμενο στάδιο, επιχειρήθηκε η διερεύνηση των άγνωστων μηχανισμών που διέπουν το δίπλωμα και την αυτοσυγκρότηση των αμυλοειδογόνων πρωτεϊνών σε ινίδια με χρήση υπολογιστικών μεθόδων δομικής βιοπληροφορικής

Exploring the 'aggregation-prone' core of human Cystatin C: A structural study.

Amyloidogenic proteins like human Cystatin C (hCC) have been shown to form dimers and oligomers by exchange of subdomains of the monomeric proteins. Normally, the hCC monomer, a low molecular type 2 Cystatin, consists of 120 amino acid residues and functions as an inhibitor of cysteine proteases. The oligomerization of hCC is involved in the pathophysiology of a rare form of amyloidosis namely Icelandic hereditary cerebral amyloid angiopathy, in which an L68Q mutant is deposited as amyloid in brain arteries of young adults. In order to find the shortest stretch responsible to drive the fibril formation of hCC, we have previously demonstrated that the LQVVR peptide forms amyloid fibrils, in vitro (Tsiolaki et al., 2015). Predictions by AMYLPRED, an amyloidogenic determinant prediction algorithm developed in our lab, led us to synthesize and experimentally study two additional predicted peptides derived from hCC. Along with our previous findings, in this work, we reveal that these peptides self-assemble, in a similar way, into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies have shown. Further to our experimental results, all three peptides seem to have a fundamental contribution in forming the ‘‘aggregation-prone’’ core of human Cystatin C

Structure-based machine-guided mapping of amyloid sequence space reveals uncharted sequence clusters with higher solubilities

An increasing number of amyloid structures are determined. Here, the authors present the structure-based amyloid core sequence prediction method Cordax that is based on machine learning and allows the detection of aggregation-prone regions with higher solubility, disorder and surface exposure, and furthermore predicts the structural topology, orientation and overall architecture of the resulting putative fibril core

Structural analysis of peptide-analogues of human Zona Pellucida ZP1 protein with amyloidogenic properties: insights into mammalian Zona Pellucida formation.

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as 'aggregation-prone' by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted 'aggregation-prone' parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization

Structure-based machine-guided mapping of amyloid sequence space reveals uncharted sequence clusters with higher solubilities

The amyloid conformation can be adopted by a variety of sequences, but the precise boundaries of amyloid sequence space are still unclear. The currently charted amyloid sequence space is strongly biased towards hydrophobic, beta-sheet prone sequences that form the core of globular proteins and by Q/N/Y rich yeast prions. Here, we took advantage of the increasing amount of high-resolution structural information on amyloid cores currently available in the protein databank to implement a machine learning approach, named Cordax (https://cordax.switchlab.org), that explores amyloid sequence beyond its current boundaries. Clustering by t-Distributed Stochastic Neighbour Embedding (t-SNE) shows how our approach resulted in an expansion away from hydrophobic amyloid sequences towards clusters of lower aliphatic content and higher charge, or regions of helical and disordered propensities. These clusters uncouple amyloid propensity from solubility representing sequence flavours compatible with surface-exposed patches in globular proteins, functional amyloids or sequences associated to liquid-liquid phase transitions.status: publishe

WALTZ-DB 2.0: an updated database containing structural information of experimentally determined amyloid-forming peptides

Transition of soluble proteins into insoluble amyloid fibrils is driven by self-propagating short sequence stretches. However, accurate prediction of aggregation determinants remains challenging. Here, we describe WALTZ-DB 2.0, an updated and significantly expanded open-access database providing information on experimentally determined amyloid-forming hexapeptide sequences (http://waltzdb.switchlab.org/). We have updated WALTZ-DB 2.0 with new entries, including: (i) experimental validation of an in-house developed dataset of 229 hexapeptides, using electron microscopy and Thioflavin-T binding assays; (ii) manual curation of 98 amyloid-forming peptides isolated from literature. Furthermore, the content has been expanded by adding novel structural information for peptide entries, including sequences of the previous version. Using a computational methodology developed in the Switch lab, we have generated 3D-models of the putative amyloid fibril cores of WALTZ-DB 2.0 entries. Structural models, coupled with information on the energetic contributions and fibril core stabilities, can be accessed through individual peptide entries. Customized filtering options for subset selections and new modelling graphical features were added to upgrade online accessibility, providing a user-friendly interface for browsing, downloading and updating. WALTZ-DB 2.0 remains the largest open-access repository for amyloid fibril formation determinants and will continue to enhance the development of new approaches focused on accurate prediction of aggregation prone sequences.status: publishe

An N-terminal pro-atrial natriuretic peptide (NT-proANP) 'aggregation-prone' segment involved in isolated atrial amyloidosis.

Isolated atrial amyloidosis (IAA) is a common localized form of amyloid deposition within the atria of the aging heart. The main constituents of amyloid fibrils are atrial natriuretic peptide (ANP) and the N-terminal part of its precursor form (NT-proANP). An ‘aggregation-prone’ heptapeptide ( 114KLRALLT120) was located within the NT-proANP sequence. This peptide self-assembles into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies reveal. Consequently, remedies/drugs designed to inhibit the aggregation tendency of this ‘aggregation-prone’ segment of NT-proANP may assist in prevention/treatment of IAA, congestive heart failure (CHF) or atrial fibrillation (AF

Hidden Aggregation Hot-Spots on Human Apolipoprotein E: A Structural Study

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aβ peptide in amyloid plaques in Alzheimer’s disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer’s disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE−Αβ complex. Our in vitro biophysical results prove that apoE peptide−analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE−Αβ complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αβ; taking an important step forward in the field of Alzheimer’s anti-aggregation drug development

X-ray diffraction patterns produced from oriented fibres of mature fibril suspensions.

<p>The mature fibrils have derived from: (a) ZPH_A peptide, (b) ZPH_G peptide, (c) a mixture of ZPH_A & ZPH_G peptides. The meridian, M (direction parallel to the fibre axis, F) is vertical and the equator, E, is horizontal in this display. All X-ray diffraction patterns are clearly “cross-β” patterns <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Geddes1" target="_blank">[71]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Jahn1" target="_blank">[73]</a>. (a) An intense meridional 4.7 Å reflection corresponds to the spacing of successive hydrogen bonded β-strands, perpendicular to the fiber axis, whereas the 9.1 Å reflection on the equator is attributed to the packing distance of β-sheets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 4). The sheets are packed parallel to the fiber axis. (b) The X-ray diffraction pattern of the ZPH_G peptide also exhibits similar reflections that indicate the presence of a “cross-β” conformation. The structural repeat of 4.7 Å corresponds to the spacing of successive β-strands arranged perpendicular to the fiber axis, while the 12.4 Å spacing on the equator, corresponds to the packing distance of consecutive β-sheet parallel to the fibre axis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 5). (c) The X-ray pattern produced from the mixture of the ZPH_A & ZPH_G peptide fibril suspensions is clearly a combination of the diffraction patterns produced by the individual fibers formed from the ZPH_A and ZPH_G peptide's fibril suspensions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 6). The 2.4 Å, 3.8 Å, 7.1 Å and 9.1 Å reflections are due to the presence of fibrils formed by the ZPH_A peptide, whereas the 12.4 Å reflection is produced by fibrils formed by the ZPH_G peptide (corresponding to β-sheet packing distance). The intense 4.7 Å reflection has contributions from both fibril populations and this is in agreement with the EM photograph of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-g002" target="_blank">Fig. 2c</a>.</p