This year’s BBRC Symposium was organized by

Igor Stagljar, University of Toronto, Local Organizer

Wolfgang Baumeister, Max Planck Institut (MPI) für Biochemie, Editor-in-Chief BBRC

Valerie Teng-Broug, Elsevier, Senior Publisher BBRC

BBRC Symposium

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ISSN 0006-291X

Biochemical and Biophysical

Research Communications

BBRC Available online at www.sciencedirect.com

ScienceDirect

Vol. 459/4 (2015) 561–720

4594 Volume 459, Issue 4, April 17, 2015

YBBRC_v459_i4_COVER.indd 1 27-03-2015 10:46:53

Friday, 7 October 2016, 9am–5pm

University of TorontoChestnut Conference Centre, 89 Chestnut Street, Toronto

Register at www.bit.ly/BBRCSymposium

BBRC Symposium

Biochemical and Biophysical Research Communications (BBRC) is a broad scope Life Sciences journal which publishes articles in a variety of topics ranging from cancer biology to computational biology and bioinformatics. Historically, the BBRC Editorial Board has comprised eminent scientists, well respected in their field and with strong peer review publications. They have dedicated their time and energy towards the betterment of science and BBRC and won many prestigious awards in their respective fields.

The BBRC Symposium is designed to showcase the diverse proficiencies of the Editorial Board and highlight the different areas of life science published in this journal. The first of these was held in May 2015 at NCBS, Bengaluru, India. The venue for this year’s BBRC Symposium is Toronto, Canada on Friday, October 7th. It will feature 3 guest speakers, Drs. Liliana Attisano, Igor Jurisica and Lewis Kay, our Editor-in-Chief, Dr. Wolfgang Baumeister, and 4 members of the Editorial Board.

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ISSN 0006-291X

Biochemical and Biophysical

Research Communications

BBRC Available online at www.sciencedirect.com

ScienceDirect

Vol. 459/4 (2015) 561–720

4594 Volume 459, Issue 4, April 17, 2015

YBBRC_v459_i4_COVER.indd 1 27-03-2015 10:46:53

Programme

Morning session chaired by Ernesto Carafoli

9:00am Welcome wordIgor Stagljar University of Toronto and BBRC Symposium organizerErnesto Carafoli University of PadovaWolfgang Baumeister Max Planck Institut (MPI) für Biochemie

9:15am Wolfgang Baumeister Max Planck Institut (MPI) für BiochemieStructural studies of the 26S proteasome ex situ and in situ

Lewis Kay University of TorontoThe importance of protein dynamics in health and disease

10:45am Coffee break

11:15am Sergio Pantano Institut Pasteur de MontevideoDesign of a universal FRET-tag reveals cAMP nano-domains at β-adrenergic cascade

Igor Jurisica University of TorontoReducing Imprecision in Precision Medicine

12:45pm Lunch break

Afternoon session chaired by Wolfgang Baumeister

1:45pm Gerald W. Hart Johns Hopkins University School of MedicineNutrient Regulation of Transcription & Signaling by O-GlcNAcylation: Fundamental Roles in Diabetes and Obesity

Naoyuki Taniguchi RIKENImplication of N-glycan in pathogenesis of Alzheimer’s disease and its possible glycan-based therapeutics

3:15pm Refreshment break

3:30pm Yong-Keun Jung Seoul National UniversityAmyloid neurotoxicity in Alzheimer’s disease

Liliana Attisano University of TorontoUncovering regulators of the Hippo pathway mediators YAP/TAZ in breast cancer cells by multidimensional screening.

5:00pm Close

Wolfgang BaumeisterMax Planck Institut (MPI) für Biochemie

Structural studies of the 26S proteasome ex situ and in situ

The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. The 2.5 MDa complex is built of 34 different subunits and comprises two subcomplexes: the 20S core where proteolysis takes place and one or two regulating particles which prepare substrates for degradation. Whereas the structure of its 20S core particle has been determined by X-ray crystallography two decades ago, the structure of the 19S regulatory particle, which recruits substrates, unfolds them, and translocates them to the core particle for degradation, has remained elusive. The molecular architecture of the 26S holocomplex was determined by an integrative approach based on data from single particle cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. Based on a 6Å structure of the Saccharomyces cerevisiae proteasome, and molecular dynamics-based flexible fitting we were able to generate a near-atomic model of the 26 holocomplex. Recently, we determined the structure of the human 26S protein at a resolution of 3.9 Å. Furthermore, by applying deep image classification to a very large data set, we defined its conformational landscape.

Electron cryotomography allows to perform structural studies of macromolecular and supramolecular structures in situ, i.e. in their functional cellular environments. We applied electron cryotomography with a new type of a phase plate, the Volta phase plate, to hippocampal neurons grown on EM grids and were able to locate 26S proteasomes with high fidelity and nanometer precision in these cells. Subtomogram, classification and averaging revealed the existence of different states of assembly and conformational states. From this we can infer the activity status of individual 26S complexes.

Lewis KayUniversity of Toronto

The importance of protein dynamics in health and disease

The p97/VCP ATPase is an essential, highly abundant protein that is conserved in eukaryotes, with central functions in diverse processes ranging from protein degradation to DNA damage repair and cell cycle control. Malfunctions of p97 have been implicated in degenerative diseases and cancer. Using solution-based Nuclear Magnetic Resonance spectroscopy that has been optimized for applications to molecular machines we have investigated how disease-causing mutations in human p97 affect inter-domain dynamics of the N-terminal domain (NTD) that binds adaptor proteins involved in controlling p97 function. Our results provide a molecular basis for understanding the mechanism of action of a class of disease causing mutations that leads to deregulation of NTD dynamics in the ADP-bound form of the enzyme, shifting the NTD conformation towards an ATP-like state and interfering with adaptor binding. An allosteric network is mapped that facilitates inter-domain communication, with potential implications for modulation of enzyme activity by drug molecules. This work will be highlighted in my presentation.

Sergio PantanoInstitut Pasteur de Montevideo

Design of a universal FRET-tag reveals cAMP nano-domains at β-adrenergic cascade

Compartmentalized cAMP/PKA signaling is a well-established paradigm with important physiological implications. However, a detailed understanding of the properties, regulation and function of local cAMP/PKA pools is still missing, mostly because of the lack of proper detection tools. Aimed to improve the 3D characterization of local signaling compartments, we used molecular simulation techniques to develop a novel FRET sensor (named CUTie) that detects compartmentalized cAMP with unprecedented spatial resolution. When targeted to multiprotein complexes located within few hundreds nanometers of each other in cardiomyocytes, CUTie reveals cAMP signals with distinct amplitude and kinetics. The nano-heterogeneity of the cAMP signal provides novel insight into fundamental mechanisms of cardiac contraction with potential relevance to pinpoint specific molecular targets for the treatment of cardiac disease.

Igor JurisicaUniversity of Toronto

Reducing Imprecision in Precision Medicine

To fathom cancer development processes, we need to systematically integrate diverse types of information, including multiple high-throughput datasets and diverse annotations. This comprehensive and integrative analysis will lead to data-driven precision medicine, and in turn will help us to develop new hypotheses, and answer complex questions such as what factors cause disease; which patients are at high risk; will patients respond to a given treatment; how to rationally select a combination therapy to individual patient, etc.

Thousands of potentially important proteins remain poorly characterized. Computational biology methods, including machine learning, data mining and visualization, can help fill this gap with accurate predictions, making disease modeling more comprehensive. Intertwining computational prediction and modeling with biological experiments will lead to more useful findings faster and more economically.

Gerald W. Hart Johns Hopkins University School of Medicine

Nutrient Regulation of Transcription & Signaling by O-GlcNAcylation: Fundamental Roles in Diabetes and Obesity

O-GlcNAcylation cycles on and off thousands of nucleocytoplasmic proteins and has extensive crosstalk with protein phosphorylation. O-GlcNAc cycles on nearly all proteins involved in transcription, where it regulates gene expression in response to nutrients. O-GlcNAc also regulates the cycling of the TATA-binding (TBP) protein on and off DNA during the transcription cycle and is required for TBP to bend DNA.

Targeted, inducible, deletion of the O-GlcNAc Transferase in CAMKII positive (excitatory) neurons of adult mice results in a morbidly obese mouse with a satiety defect. Thus, O-GlcNAcylation not only serves as a nutrient sensor in all cells, but also is directly involved in appetite regulation. O-GlcNAcylation also plays an important role in the trafficking of the AMPA receptors in neurons and in the development of functional synaptic spines. Recent studies have shown that more than one-half of all human protein kinases are modified by O-GlcNAc and all kinases that have been tested are indeed regulated in some way by the sugar. Abnormal O-GlcNAcylation of CAMKII contributes directly to diabetic cardiomyopathy and to arrhythmias associated with diabetes. Prolonged elevation of O-GlcNAc, as occurs in diabetes, contributes directly to diabetic complications and is a major mechanism of glucose toxicity. Supported by NIH P01HL107153, R01DK61671 and N01-HV-00240. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.

Naoyuki TaniguchiRIKEN

Implication of N-glycan in pathogenesis of Alzheimer’s disease and its possible glycan-based therapeutics

Glycosyltransferases involved in N-glycan biosynthesis are implicated in various diseases such as cancer, COPD and Alzheimer’s disease (AD).1

AD is the most common neurodegenerative disease and is a progressive disease that destroys memory and other important mental function. Nearly 44 million people have AD or a related dementia worldwide. It is well known that neurofibrillary tangles (NFT) and amyloid plaques are hallmarks of AD. Our group has been interested in various glycosyltransferases involved in the biosynthesis of N-glycan branching and their implication for the disease.

The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer’s disease (AD). We found that BACE1 is modified with bisecting GlcNAc, a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less co-localized with APP, leading to accelerated lysosomal degradation. The effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD. We have developed a high through put screaning method for inhibitors against GnT-III and found several candidate molecules.

References 1.  Taniguchi N & Kizuka Y (2015) Glycans and cancer: role of N-glycans in cancer biomarker, progression and metastasis, and therapeutics. Adv Cancer Res126:11-51.

2.  Kizuka Y. et al. (2015) An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer’s disease.  EMBO Mol Med 7(2):175-189

Yong-Keun JungSeoul National University

Amyloid neurotoxicity in Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by the progressive loss of memory and the neuronal degeneration. The pathological hallmarks of AD are the presence of senile plaques consisting of amyloid beta (Ab) peptide and neurofibrillary tangles (NFT) formed by abnormally hyperphosphorylated tau. The pathological features of AD, including the memory loss and neuronal degeneration, are believed to be associated with the accumulation of Ab in the brain. We found that Fcg-receptor IIb (FcgRIIb) mediates Ab  neurotoxicity and neurodegeneration. FcgRIIb is significantly up-regulated in the hippocampus of AD brains and neuronal cells exposed to Ab1-42. Neuronal FcgRIIb activates ER stress and caspase-12, and FcgRIIb knockout primary neurons are resistant to Ab1-42-induced cell deathin vitro. FcgRIIb deficiency ameliorates Ab1-42-induced inhibition of long-term potentiation and inhibits the reduction of synaptic density by naturally secreted Ab. Moreover, genetic depletion of FcgRIIb rescues the memory impairments in AD model mice. In an action mode of FcgRIIb in Ab neurotoxicity, we found that soluble Ab1-42 oligomers interact with FcgRIIb in vitro and in AD brains, and inhibition of their interaction blocks Ab1-42 neurotoxicity. Thus, we conclude that FcgRIIb has an aberrant but essential role in Ab1-42-mediated neuronal dysfunction. Moreover, we found that the FcgRIIb-SHIP2 axis is critical in Ab1-42-induced tau pathology. Fcgr2b knockout or antagonistic FcgRIIb antibody inhibited Ab1-42-induced tau hyperphosphorylation and rescued memory impairments in AD mouse models. FcgRIIb phosphorylation at Tyr273 was found in AD brains, in neuronal cells exposed to Ab1-42, and recruited SHIP2 to form a protein complex. Consequently, treatment with Ab1-42 increased PtdIns(3,4)P2 levels from PtdIns(3,4,5)P3 to mediate tau hyperphosphorylation. Further, we found that targeting SHIP2 expression by lentiviral siRNA in 3xTg-AD mice or pharmacological inhibition of SHIP2 potently rescued tau hyperphosphorylation and memory impairments. Thus, we concluded that the FcgRIIb-SHIP2 axis links Ab neurotoxicity to tau pathology, providing insight into therapeutic potential against AD

Liliana AttisanoUniversity of Toronto

Uncovering regulators of the Hippo pathway mediators YAP/TAZ in breast cancer cells by multidimensional screening

The Hippo signalling pathway is a key regulator of tissue growth and organogenesis.  The pathway is comprised of a core MST/LATS kinase cassette that phosphorylates and promotes cytoplasmic localization of the transcriptional regulators, TAZ and YAP. To uncover novel Hippo pathway regulators, we conducted multidimensional high throughput screens involving LUMIER-based protein-protein interaction mapping, functional cDNA overexpression and siRNA knockdown assays using TAZ/YAP transcriptional reporters and imaging-based subcellular localization.  These efforts have uncovered several kinases that function to block Hippo pathway activity, such as members of the MARK kinase family.  We have observed that MARKs function to inhibit the Hippo pathway by directly phosphorylating components of the kinase cassette and disrupting its activity.  This serves to promote TAZ/YAP transcriptional activity and enhances the growth and migration in breast cancer cells.  Our screens also uncovered several positive Hippo/TAZ/YAP regulators such as ARHGEF7, commonly known as βetaPIX, a multidomain-containing guanine nucleotide exchange factor (GEF). We have shown that the betaPIX binds both LATS and TAZ/YAP to promote LATS-mediated repression of TAZ/YAP. βetaPIX functions downstream of both high cell density and actin cytoskeletal rearrangements and loss of betaPIX expression in normal mammary epithelial cells strongly reduces TAZ/YAP phosphorylation, promotes nuclear localization and increases target gene expression.  Increased expression of βetaPIX, in a triple negative breast cancer cell line, which displays constitutively nuclear TAZ/YAP, recouples the Hippo kinase cassette to the regulation of TAZ/YAP activity. Thus, our screens have identified both positive and negative regulators of the Hippo pathway that function in normal and tumorigenic breast cancer cell lines.

For the full aims & scope, instructions for authors and Editorial Board details, please visit the journal homepage: elsevier.com/locate/ybbrc

Supports Open Access

Bioch

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ISSN 0006-291X

Biochemical and Biophysical

Research Communications

BBRC Available online at www.sciencedirect.com

ScienceDirect

Vol. 459/4 (2015) 561–720

4594 Volume 459, Issue 4, April 17, 2015

YBBRC_v459_i4_COVER.indd 1 27-03-2015 10:46:53