Hilal Lashuel

Publications (84) View all

• Article: Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.

  Farah El-Turk, Bruno Fauvet, Amer Ashrafi, Hajer Ouertatani-Sakouhi, Min-Kyu Cho, Marilisa Neri, Michele Cascella, Ursula Rothlisberger, Florence Pojer, Markus Zweckstetter, Hilal Lashuel
  [show abstract] [hide abstract]
  ABSTRACT: Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.
  PLoS ONE 01/2012; 7(9):e45024. · 4.09 Impact Factor
• Article: The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor.

  Farah El-Turk, Michele Cascella, Hajer Ouertatani-Sakouhi, Raghavendran Lakshmi Narayanan, Lin Leng, Richard Bucala, Markus Zweckstetter, Ursula Rothlisberger, Hilal A Lashuel
  [show abstract] [hide abstract]
  ABSTRACT: Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.
  Biochemistry 10/2008; 47(40):10740-56. · 3.42 Impact Factor
• Article: The size of the proteasomal substrate determines whether its degradation will be mediated by mono- or polyubiquitylation.

  Nitzan Shabek, Yifat Herman-Bachinsky, Samuel Buchsbaum, Oded Lewinson, Mahmood Haj-Yahya, Mirva Hejjaoui, Hilal A Lashuel, Thomas Sommer, Ashraf Brik, Aaron Ciechanover
  [show abstract] [hide abstract]
  ABSTRACT: A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to ∼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.
  Molecular cell 08/2012; 48(1):87-97. · 14.61 Impact Factor
• Article: Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation.

  Heiko Kroth, Annalisa Ansaloni, Yvan Varisco, Asad Jan, Nampally Sreenivasachary, Nasrollah Rezaei-Ghaleh, Valérie Giriens, Sophie Lohmann, María Pilar López-Deber, Oskar Adolfsson, Maria Pihlgren, Paolo Paganetti, Wolfgang Froestl, Luitgard Nagel-Steger, Dieter Willbold, Thomas Schrader, Markus Zweckstetter, Andrea Pfeifer, Hilal A Lashuel, Andreas Muhs
  [show abstract] [hide abstract]
  ABSTRACT: Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.
  Journal of Biological Chemistry 08/2012; 287(41):34786-800. · 4.77 Impact Factor
• Source

  Available from: Bruno Fauvet

  Article: Phosphorylation of α-Synuclein at Y125 and S129 Alters Its Metal Binding Properties: Implications for Understanding the Role of α-Synuclein in the Pathogenesis of Parkinson's Disease and Related Disorders.

  Yu Lu, Michel Prudent, Bruno Fauvet, Hilal A Lashuel, Hubert H Girault
  [show abstract] [hide abstract]
  ABSTRACT: α-Synuclein (α-syn) is a 140-amino acid protein that plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. However, the molecular determinants that are responsible for triggering and/or propagating α-syn aggregation and toxicity remain poorly understood. Several studies have suggested that there are direct interactions between different metals and α-syn, but the role of metal ions and α-syn in the pathogenesis of PD is not firmly established. Interestingly, the majority of disease-associated post-translational modifications (PTMs) (e.g., truncation, phosphorylation, and nitration) of α-syn occur at residues within the C-terminal region (Y125, S129, Y133, and Y136) and in very close proximity to the putative metal binding sites. Therefore, we hypothesized that phosphorylation within this domain could influence the α-syn-metal interactions. In this paper, we sought to map the interactions between the di- and trivalent cations, Cu(II), Pb(II), Fe(II), and Fe(III), and the C-terminal region of α-syn encompassing residues 107-140 and to determine how phosphorylation at S129 or Y125 alters the specificity and binding affinity of metals using electrospray ionization-mass spectrometry (ESI-MS) and fluorescence spectroscopy. We demonstrate that D115-M116 and P128-S129 act as additional Cu(II) binding sites and show for the first time that the residues P128-S129 and D119 are also involved in Pb(II) and Fe(II) coordination, although D119 is not essential for binding to Fe(II) and Pb(II). Furthermore, we demonstrate that phosphorylation at either Y125 or S129 increases the binding affinity of Cu(II), Pb(II), and Fe(II), but not Fe(III). Additionally, we also show that phosphorylations at these residues lead to a shift in the binding sites of metal ions from the N-terminus to the C-teminus. Together, our findings provide critical insight into and expand our understanding of the molecular and structural bases underlying the interactions between α-syn and metal ions, including the identification of novel metal binding sites, and highlight the potential importance of cross-talk between post-translational modifications and metal ion binding in modulating α-syn functional and aggregation properties that are regulated by its C-terminal domain.
  ACS Chemical Neuroscience 11/2011; 2(11):667-75. · 3.68 Impact Factor