Marko

Tuning liver pyruvate kinase activity up or down with a new class of allosteric modulators

Amalyn Nain-Perez , Oscar Nilsson , Aleksei Lulla , Liliana Håversen, Paul Brear, Sara Liljenberg , Marko Hyvönen, Jan Borén, Morten Grøtli

European Journal of Medicinal Chemistry, 15:250:115177 (2023)
DOI: 10.1016/j.ejmech.2023.115177
Pubmed: 36753880
PDB coordinates:
7FRW (3D view ), 7FRV (3D view ), 7FRX (3D view ), 7FRZ (3D view ), 7FS0 (3D view ), 7FS1 (3D view ), 7FS2 (3D view ), 7FS3 (3D view ), 7FS4 (3D view ), 7FS5 (3D view ), 7FS6 (3D view ), 7FS7 (3D view ), 7FS8 (3D view ), 7FS9 (3D view ), 7FSA (3D view ), 7FSB (3D view ), 7FSC (3D view ), 7FSD (3D view )

Abstract

The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 μM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Continue reading →

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Bruce Printsteen

Not sure if there was a proper consultation on this, but the 3D printer has a name now.

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Going 3D, really 3D

After 20+ years of determining atomic level structures of proteins in three dimensions, we are going properly 3D. Our Prusa i3 MKS3+ has arrived!

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Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture

Philipp KnyphausenMariana Rangel PereiraPaul Brear, Marko HyvönenLutz JermutusFlorian Hollfelder

Nature Communications 14:768 (2023)
DOI: 10.1038/s41467-023-36099-7
Pubmed: 36765057
PDB coordinates:6YV5 (3D view ), 6YV6 (3D view )

Abstract

Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. Continue reading →

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One of those weeks

HPLC plays up with random UV peaks, fraction collector tries to fail its calibration, centrifuge bottles crack with half of mammalian expression culture lost, fluorescent detector cuts off randomly.

And it’s only a Wednesday.

Panton Arms, here we come.

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Part II projectiles have arrived

Welcome to Sarah and Bocheng for joining the group for their part II projects (third year undergraduate project in normal terminology), under the supervision of Shiv and Emma, respectively.

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Microfluidics-enabled fluorescence-activated cell sorting of single pathogen-specific antibody secreting cells for the rapid discovery of monoclonal antibodies

Katrin Fischer, Aleksei Lulla, Tsz Y So, Pehuén Pereyra-Gerber, Matthew I. J. Raybould, Timo N. Kohler, Tomasz S. Kaminski, Juan Carlos Yam-Puc, Robert Hughes, Florian Leiß-Maier, Paul Brear, Nicholas J. Matheson,Charlotte M. Deane, Marko Hyvönen, James E. D. Thaventhiran, Florian Hollfelder

BioRxiv, posted 12 Jan 2023
DOI: 10.1101/2023.01.10.523494
PDB coordinates: 8BE1 (3D view)
Plasmids in Addgene: pExp-His-ZBasic-RBD, pExp-His-ZBasic-RBD-Avi

Abstract

Monoclonal antibodies are increasingly used to prevent and treat viral infections, playing a pivotal role in pandemic response efforts. Antibody secreting cells (ASCs, plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methodologies to study antigen-specific ASCs either have low throughput, require expensive and labour-intensive screening or are technically demanding and therefore not accessible to the wider research community. Here, we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs: we combine microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. Continue reading →

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A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

Jerome Jatzlau, Wiktor Burdzinski, Michael Trumpp, Leon Obendorf, Kilian Roßmann, Katharina Ravn, Marko Hyvönen, Francesca Bottanelli, Johannes Broichhagen & Petra Knaus 

Communications Biology 6: 34 (2023)
DOI: 10.1038/s42003-022-04388-4
Pubmed: 36635368

Abstract

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.

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Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

Umberto Maria Battisti, Chunxia Gao, Oscar Nilsson, Fady Akladios, Aleksei Lulla, Agnieszka Bogucka, Amalyn Nain-Perez, Liliana Håversen, Woonghee Kim, Jan Boren, Marko Hyvönen, Mathias Uhlen, Adil Mardinoglu, Morten Grøtli

ChemBioChem, accepted article 17th Oct (2022)
DOI: 0.1002/cbic.202200339
Pubmed: 36250581

Abstract

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 = 0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

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Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α

Paul Brear and Marko Hyvönen

Acta Crystallographica section F 78: 348-353 (2022)
DOI: 10.1107/S2053230X22008767

PDB coordinates:
7z39 (3D view)

Abstract

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Continue reading →

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