S100A9 EXERTS INSULIN-INDEPENDENT ANTI-DIABETIC AND ANTI-INFLAMMATORY EFFECTS
Science advances; January 5th, 2024
Here, we report an insulin signaling–independent pathway able to improve glycemic control in rodents. Co-treatment with recombinant S100 calcium-binding protein A9 (S100A9) enabled increased adherence to glycemic targets with half as much insulin and without causing hypoglycemia. Mechanistically, we demonstrate that the hyperglycemia-suppressing action of S100A9 is due to a Toll-like receptor 4–dependent increase in glucose uptake in specific skeletal muscles (i.e., soleus and diaphragm).In addition, we found that T1DM mice have abnormal systemic inflammation, which is resolved by S100A9 therapy alone (or in combination with low insulin), hence uncovering a potent anti-inflammatory action of S100A9 in T1DM.
HEPATIC NON-PARENCHYMAL S100A9-TLR4-MTORC1 AXIS NORMALIZES DIABETIC KETOGENESIS
Nature Communications; July 15th, 2022
In a collaborative effort, together with the Coppari group at UNIGE, we report an insulin-independent pathway able to normalize diabetic ketogenesis and show that recombinant S100A9 administration restrains ketogenesis and improves hyperglycemia without causing hypoglycemia in diabetic mice. Anna Höfler from our group expressed and purified recombinant mouse and human S100A9.
STRUCTURAL BASIS OF HUMAN SEPARASE REGULATION BY SECURIN AND CDK1-CYCLIN B1
Nature; August 11th, 2021
We used cryoEM to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, identified a novel phosphate-binding pocket in B-type cyclins and explains the mutual inhibition of separase and CDK1 when forming a complex. This work was in collaboration with the labs of Stefan Raunser and Dave Morgan.
BIPARTITE BINDING AND PARTIAL INHIBITION LINKS DEPTOR AND MTOR IN A MUTUALLY ANTAGONISTIC EMBRACE
eLife; September 14th, 2021
DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.
STRUCTURE OF THE DOCK2−ELMO1 COMPLEX PROVIDES INSIGHTS INTO REGULATION OF THE AUTO-INHIBITED STATE
Nature Communications; July 10th, 2020
DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. We determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition exposes binding sites for RAC1, and RHOG and BAI GPCRs on ELMO1. Our structure rationalises how up-stream effectors destabilise the auto-inhibited state to promote an active GEF.
A TRI-IONIC ANCHOR MECHANISM DRIVES UBE2N-SPECIFIC RECRUITMENT AND K63-CHAIN UBIQUITINATION IN TRIM LIGASES
Nature Communications; October 10th, 2019
TRIM21 ubiquitination activity is dependent on formation of K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. We reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other related enzymes. A tri-ionic motif provides optimal anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. The same mechanism is employed by TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif provides a generic E2-specific catalysis mechanism for RING E3s.
THE CRYOEM STRUCTURE OF THE SACCHAROMYCES CEREVISIAE RIBOSOME MATURATION FACTOR REA1
eLife; November 21st, 2018
pre-60S particles mature by transiently interacting with various assembly factors. The ATPase Rea1 generates force to remove assembly factors from pre-60S particles. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. We reveal the architecture of the Rea1 linker that is involved in force generation and extends from the AAA+ ring.
CRYO-EM STRUCTURE OF A METAZOAN SEPARASE-SECURIN COMPLEX AT NEAR-ATOMIC RESOLUTION
Nature Structural & Molecular Biology; August 17th, 2017
Our analyses provide insight into the overall architecture of separase, explain the substrate-occlusion inhibitory mechanism of securin, and rationalize the strict necessity of an arginine residue in the P1 binding pocket to mediate substrate-assisted cleavage. We also demonstrate the applicability of cryo-EM for the resolution of structures of macromolecules ~150 kDa in size that are difficult to crystallize.
FAST NATIVE-SAD PHASING FOR ROUTINE MACROMOLECULAR STRUCTURE DETERMINATION
Nature Methods; December 15th, 2014
We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.
A DDX6-CNOT1 COMPLEX AND W-BINDING POCKETS IN CNOT9 REVEAL DIRECT LINKS BETWEEN MIRNA TARGET RECOGNITION AND SILENCING
Molecular Cell; June 5th, 2014
CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. Our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.
STRUCTURE AND ASSEMBLY OF THE NOT MODULE OF THE HUMAN CCR4-NOT COMPLEX
Nature Structural & Molecular Biology; October 13th, 2013
The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.
STRUCTURE OF THE PAN3 PSEUDOKINASE REVEALS THE BASIS FOR INTERACTIONS WITH THE PAN2 DEADENYLASE AND THE GW182 PROTEINS
Molecular Cell; August 8th, 2013
The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 structure that, unexpectedly, forms intertwined and asymmetric homodimers. The most remarkable feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.
A DIRECT INTERACTION BETWEEN DCP1 AND XRN1 COUPLES MRNA DECAPPING TO 5' EXONUCLEOLYTIC DEGRADATION
Nature Structural & Molecular Biology; November 11th, 2012
The removal of the mRNA 5′ cap structure by the decapping enzyme DCP2 leads to rapid 5′→3′ mRNA degradation by XRN1. DCP2 associates with the decapping activators EDC4 and DCP1. Here we show that XRN1 directly interacts with EDC4 and DCP1. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. Our findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5′→3′ mRNA degradation.
CRYSTAL STRUCTURE OF THE MID-PIWI LOBE OF A EUKARYOTIC ARGONAUTE PROTEIN
Proceedings of the National Academy of Sciences; June 6th, 2011
Argonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. We present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5' end, and residues from both domains contribute to binding.
CRYSTAL STRUCTURE AND LIGAND BINDING OF THE MID DOMAIN OF A EUKARYOTIC ARGONAUTE PROTEIN
EMBO reports; June 11th, 2010
Argonaute (AGO) proteins are core components of RNA-induced silencing complexes and have essential roles in RNA-mediated gene silencing. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE-2 from Neurospora crassa. This domain adopts a Rossmann-like fold and recognizes the 5′-terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′-nucleotide-binding site shares common residues with a second, adjacent ligand-binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.