Guillaume Lamour

Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes

Because of their pervasiveness in eukaryotic genomes and their unique properties, understanding the role that ID (intrinsically disordered) regions in proteins play in the interactome is essential for gaining a better understanding of the network. Especially critical in determining this role is their ability to bind more than one partner using the same region. Studies have revealed that proteins containing ID regions tend to take a central role in protein interaction networks; specifically, they act as hubs, interacting with multiple different partners across time and space, allowing for the co-ordination of many cellular activities. There appear to be three different modules within ID regions responsible for their functionally promiscuous behaviour: MoRFs (molecular recognition features), SLiMs (small linear motifs) and LCRs (low complexity regions). These regions allow for functionality such as engaging in the formation of dynamic heteromeric structures which can serve to increase local activity of an enzyme or store a collection of functionally related molecules for later use. However, the use of promiscuity does not come without a cost: a number of diseases that have been associated with ID-containing proteins seem to be caused by undesirable interactions occurring upon altered expression of the ID-containing protein.

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

Background: Collagen macromolecules are biologically relevant substrates in tissue remodeling and bone-related diseases. Results: We investigated the action of cysteine proteases on the structural integrity and mechanical functionality of collagen fibers. Conclusion: Using ultrastructural and biochemical techniques, we present a model of collagen fiber degradation via cathepsin K. Significance: Our data provide new insights in matrix degradation and may allow new strategies to inhibit it. Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Youngs moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.

The prion protein ligand, stress-inducible phosphoprotein 1, regulates amyloid-β oligomer toxicity

In Alzheimers disease (AD), soluble amyloid-β oligomers (AβOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrPC). However, it is unknown whether other ligands of PrPC can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrPC in the vicinity of the AβO binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in AβO toxicity. We confirmed the specific binding of AβOs and STI1 to the PrP and showed that STI1 efficiently inhibited AβO binding to PrP in vitro (IC50 of ∼70 nm) and also decreased AβO binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented AβO-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to AβO-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both AβO binding to PrPC and PrPC-dependent AβO toxicity were inhibited by TPR2A, the PrPC-interacting domain of STI1. Additionally, PrPC–STI1 engagement activated α7 nicotinic acetylcholine receptors, which participated in neuroprotection against AβO-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrPC ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset AβO-induced toxicity.

High intrinsic mechanical flexibility of mouse prion nanofibrils revealed by measurements of axial and radial Young's moduli.

Self-templated protein aggregation and intracerebral deposition of aggregates, sometimes in the form of amyloid fibrils, is a hallmark of mammalian prion diseases. What distinguishes amyloid fibrils formed by prions from those formed by other proteins is not clear. On the basis of previous studies on yeast prions that correlated high intrinsic fragmentation rates of fibrils with prion propagation efficiency, it has been hypothesized that the nanomechanical properties of prion amyloid such as strength and elastic modulus may be the distinguishing feature. Here, we reveal that fibrils formed by mammalian prions are relatively soft and clearly in a different class of rigidities when compared to nanofibrils formed by nonprions. We found that amyloid fibrils made of both wild-type and mutant mouse recombinant PrP(23-231) have remarkably low axial elastic moduli of 0.1-1.4 GPa. We demonstrate that even the proteinase K resistant core of these fibrils has similarly low intrinsic rigidities. Using a new mode of atomic force microscopy called AM-FM mode, we estimated the radial modulus of PrP fibrils at ∼0.6 GPa, consistent with the axial moduli derived by using an ensemble method. Our results have far-reaching implications for the understanding of protein-based infectivity and the design of amyloid biomaterials.

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

Background: Extracellular matrix (ECM) alterations during aging contribute to various pathological phenotypes. Results: Collagen fibrils, fibers, and bone alter their structural integrity and susceptibility toward degradation by cathepsin K when age-modified. Conclusion: Age-related modifications of collagen affect its biomechanics and proteolytic stability. Significance: Our research reveals how matrix modifications may increase the risk of ECM disorders. During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Youngs modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging.

Easyworm: an open-source software tool to determine the mechanical properties of worm-like chains

BackgroundA growing spectrum of applications for natural and synthetic polymers, whether in industry or for biomedical research, demands for fast and universally applicable tools to determine the mechanical properties of very diverse polymers. To date, determining these properties is the privilege of a limited circle of biophysicists and engineers with appropriate technical skills.FindingsEasyworm is a user-friendly software suite coded in MATLAB that simplifies the image analysis of individual polymeric chains and the extraction of the mechanical properties of these chains. Easyworm contains a comprehensive set of tools that, amongst others, allow the persistence length of single chains and the Young’s modulus of elasticity to be calculated in multiple ways from images of polymers obtained by a variety of techniques (e.g. atomic force microscopy, electron, contrast-phase, or epifluorescence microscopy).ConclusionsEasyworm thus provides a simple and efficient tool for specialists and non-specialists alike to solve a common problem in (bio)polymer science. Stand-alone executables and shell scripts are provided along with source code for further development.

Mechanically Tightening a Protein Slipknot into a Trefoil Knot

The knotted/slipknotted polypeptide chain is one of the most surprising topological features found in certain proteins. Understanding how knotted/slipknotted proteins overcome the topological difficulty during the folding process has become a challenging problem. By stretching a knotted/slipknotted protein, it is possible to untie or tighten a knotted polypeptide and even convert a slipknot to a true knot. Here, we use single molecule force spectroscopy as well as steered molecular dynamics (SMD) simulations to investigate how the slipknotted protein AFV3-109 is transformed into a tightened trefoil knot by applied pulling force. Our results show that by pulling the N-terminus and the threaded loop of AFV3-109, the protein can be unfolded via multiple pathways and the slipknot can be transformed into a tightened trefoil knot involving ∼13 amino acid residues as the polypeptide chain is apparently shortened by ∼4.7 nm. The SMD simulation results are largely consistent with our experimental findings, providing a plausible and detailed molecular mechanism of mechanical unfolding and knot tightening of AFV3-109. These simulations reveal that interactions between shearing β-strands on the threaded and knotting loops provide high mechanical resistance during mechanical unfolding.

The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein GB1

Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use. Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for designing elastomeric proteins with tailored mechanical properties.

Construction and Characterization of Kilobasepair Densely Labeled Peptide-DNA

Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.

Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils

Amyloids are fibrillar nanostructures of proteins that are assembled in several physiological processes in human cells (e.g., hormone storage) but also during the course of infectious (prion) and noninfectious (nonprion) diseases such as Creutzfeldt-Jakob and Alzheimers diseases, respectively. How the amyloid state, a state accessible to all proteins and peptides, can be exploited for functional purposes but also have detrimental effects remains to be determined. Here, we measure the nanomechanical properties of different amyloids and link them to features found in their structure models. Specifically, we use shape fluctuation analysis and sonication-induced scission in combination with full-atom molecular dynamics simulations to reveal that the amyloid fibrils of the mammalian prion protein PrP are mechanically unstable, most likely due to a very low hydrogen bond density in the fibril structure. Interestingly, amyloid fibrils formed by HET-s, a fungal protein that can confer functional prion behavior, have a much higher Youngs modulus and tensile strength than those of PrP, i.e., they are much stiffer and stronger due to a tighter packing in the fibril structure. By contrast, amyloids of the proteins RIP1/RIP3 that have been shown to be of functional use in human cells are significantly stiffer than PrP fibrils but have comparable tensile strength. Our study demonstrates that amyloids are biomaterials with a broad range of nanomechanical properties, and we provide further support for the strong link between nanomechanics and β-sheet characteristics in the amyloid core.