Biology subjects

Hebert, H.

Publications and source records attributed to Hebert, H..

8 recordsLinked to original sources

bioRxiv2023

The membrane-cytoplasmic linker defines activity of FtsH proteases

Pandemic Pseudomonas aeruginosa clone C strains encode a xenolog of FtsH (PaFtsH2), an inner-membrane associated ATP-dependent protease. FtsH1 supports growth and intrinsic antibiotic resistance but cannot be replaced by ftsH2. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Swapping residues of a short MC peptide that links transmembrane helix-2 with the cytosolic AAA+ ATPase module from PaFtsH1 into PaFtsH2 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. FtsH1 MC peptides are glycine rich. Introducing three glycines into the membrane-proximal end of PaFtsH2s MC linker is sufficient to elevate activity in vitro and in vivo. Electron microscopy including PaFtsH2 indicates that MC linker identity influences FtsH flexibility. Our findings establish that the efficiency of substrate processing by two PaFtsH isoforms depends on how they are attached to the membrane and suggest that greater linker flexibility/length allows FtsH to degrade a wider spectrum of substrates. As FtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is not a latent enzyme, rather recognizes specific substrates or is activated in specific contexts or biological niches. We hypothesize that such linkers might play a more determinative role in functionality and physiological impact of FtsH proteases than previously thought.

biochemistry↗

bioRxiv2021

Molecular chaperone ability to inhibit amyloid-derived neurotoxicity, but not amorphous protein aggregation, depends on a conserved pH-sensitive Asp residue

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but the respective mechanisms are not fully understood. The BRICHOS domain constitutes a disease-associated small heat shock protein-like chaperone family, with activities against both amyloid toxicity and amorphous protein aggregation. Here, we show that the activity of two BRICHOS domain families against Alzheimers disease associated amyloid-{beta} neurotoxicity to mouse hippocampi in vitro depends on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The conserved Asp in its ionized state promotes structural flexibility of the BRICHOS domain and has a pKa value between pH 6.0-7.0, suggesting that chaperone effects against amyloid toxicity can be affected by physiological pH variations. Finally, the Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families and animal species, indicating independent evolution of molecular chaperone activities against amyloid fibril formation and non-fibrillar amorphous protein aggregation.

biochemistry↗

bioRxiv2020

CHD7 interacts with the nucleosome acidic patch for its efficient activity via its N-terminal region

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed integrative biophysical analysis on CHD7 chromatin remodeler using crosslinking-mass spectrometry (XL-MS), cryo-Electron Microscopy (cryo-EM) and single-molecule Forster Resonance Energy Transfer (smFRET). We uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. The cryo-EM analysis on the N-CRD_Chromodomain bound to nucleosome reveals that the N-CRD interacts with the acidic patch of nucleosome. Furthermore, smFRET analysis shows the mutations in the N-CRD result in slow or highly-fluctuating remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

biochemistry↗

bioRxiv2020

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

biochemistry↗

bioRxiv2019

The polyQ expansion modulates the configuration and phosphorylation of huntingtin

The polyQ-expansion at the N-terminus of huntingtin (HTT) is the prime cause of Huntingtons disease. The recent cryo-EM structure of HTT with HAP40 provides information on the proteins prominent HEAT-repeats. Here, we present analyses of the impact of polyQ-length on the conformation of HTT by cryo-EM, the domain-interactions by cross-linking mass spectrometry and the phosphorylation of HTT. The cryo-EM analysis of normal (Q23-) and disease (Q78-) type HTTs in their apo forms shows that the structures of apo HTTs significantly differ from the structure of HTT-HAP40, and that the polyQ expansion induces global structural changes consisting of significant domain movements of the C-HEAT domain relative to the N-HEAT domain. In addition, we show that the polyQ-expansion alters the phosphorylation pattern across the full-length HTT and that the specific phosphorylation (Ser2116p) in turn affects the global structure of HTT, which influences the activity of polyQ-expanded HTT. These results provide a molecular basis for the effect of the N-terminal polyQ segment on HTT structure and activity, that may be important for the cell-selective toxicity of mutant HTT.

neuroscience↗

bioRxiv2018

Structural basis of recognition and destabilization of histone H2B ubiquitinated nucleosome by DOT1L histone H3 Lys79 methyltransferase

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B-ubiquitination. Here, we present cryo-EM structures of DOT1L complex with unmodified and H2B-ubiquitinated nucleosomes, showing that DOT1L recognizes H2B-ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L respectively. Furthermore, the structures combined with single-molecule FRET experiment show that H2B-ubiquitination enhances a non-catalytic function of DOT1L destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

biophysics↗

bioRxiv2017

A genome-wide association study finds novel genetic associations with broadly-defined headache in UK Biobank (N = 223,773)

Headache is the most common neurological symptom and a leading cause of years lived with disability. We sought to identify the genetic variants associated with a broadly-defined headache phenotype in 223,773 subjects from the UK Biobank cohort. We defined headache based on a specific question answered by the UK Biobank participants. We performed a genome-wide association study of headache as a single entity, using 74,461 cases and 149,312 controls. We identified 3,343 SNPs which reached the genome-wide significance level of P < 5 x 10-8. The SNPs were located in 28 loci, with the top SNP of rs11172113 in the LRP1 gene having a P value of 4.92 x 10-47. Of the 28 loci, 14 have previously been associated with migraine. Among 14 new loci, rs77804065 with a P value of 5.87 x 10-15 in the LINC02210-CRHR1 gene was the top SNP.\n\nPositive relationships (P < 0.001) between multiple brain tissues and genetic associations were identified through tissue expression analysis, whereas no vascular related tissues showed significant relationships. We identified several significant positive genetic correlations between headache and other psychological traits including neuroticism, depressive symptoms, insomnia, and major depressive disorder.\n\nOur results suggest that brain function is closely related to broadly-defined headache. In addition, we also found that many psychological traits have genetic correlations with headache.

genetics↗

bioRxiv2017

Structural insights into the architecture of human Importin4_histone H3/H4_Asf1a complex and its histone H3 tail binding

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The XL-MS data showed that the C-terminal region of Importin4 interacts extensively with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic path in Importin4 accommodates the histone H3 tail and that histone H3 Lys14 is the primary residue interacting with Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.

biochemistry↗