Biology subjects

Morgan, T. E.

Publications and source records attributed to Morgan, T. E..

4 recordsLinked to original sources

bioRxiv2023

KIF1C activates and extends dynein movement through the FHF cargo adaptor

Cellular cargos move bidirectionally on microtubules due to the presence of opposite polarity motors dynein and kinesin. Many studies show these motors are co-dependent, whereby one requires the activity of the other, although the mechanism is unknown. Here, using in vitro motility assays, we show that the kinesin-3 KIF1C acts both as an activator and a processivity factor for dynein. Activation only requires a fragment of the non-motor tail of KIF1C (KIF1C-stalk) to bind the cargo adaptor HOOK3. Cryo-EM, crosslinking mass spectrometry and AlphaFold2 predictions reveal this binding site to be separate from that of two constitutive factors (FTS and FHIP), which link HOOK3 to small G-proteins on cargos. We provide a structural model for how the FTS-HOOK3-FHIP1B (FHF) complex is auto-inhibited and explain how the KIF1C-stalk relieves this inhibition. Collectively, our work provides a molecular explanation for co-dependency by revealing that the mutual activation of dynein and kinesin is mediated through their shared adaptor. Many adaptors bind both dynein and kinesins, suggesting this mechanism could be generalised to other bidirectional complexes.

cell biology↗

bioRxiv2023

Alzheimer's Disease associations of ferritin and glutathione with oxidative damage and neuronal loss

ABSTRACTIron-mediated cell death (ferroptosis) is a proposed mechanism of Alzheimers disease (AD) pathology. While iron is essential for basic biological functions, its reactivity generates oxidants which contribute to cell damage and death. To further resolve mechanisms of iron-mediated toxicity in AD, we analyzed postmortem human brain and ApoEFAD mice. AD brains had decreased antioxidant enzymes, including those mediated by glutathione (GSH). Subcellular analyses of AD brains showed greater oxidative damage and lower antioxidant enzymes in lipid rafts, the site of amyloid processing, than in the non-raft membrane fraction. ApoE4 carriers had lower lipid raft yield with greater membrane oxidation. The hypothesized role of iron to AD pathology was tested in ApoEFAD mice by iron chelation with deferoxamine, which decreased fibrillar amyloid and lipid peroxidation, together with increased GSH-mediated antioxidants. These novel molecular pathways in iron mediated damage during AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/534324v3_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1a28ddborg.highwire.dtl.DTLVardef@10f57f7org.highwire.dtl.DTLVardef@583c8corg.highwire.dtl.DTLVardef@ef45f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Hypothesis: AD brain lipid peroxidation is driven by increased brain iron and decreased antioxidant defenses. Schema shows proteins that mediate iron metabolism in relation to lipid peroxidation (HNE) and antioxidant defenses in prefrontal cortex. AD-associated increase (red), decrease (blue), or no change (grey), relative to cognitively normal elderly controls. A{beta}; amyloid beta, ALDH2; alcohol dehydrogenase, APP; amyloid precursor protein, DMT1; divalent metal transporter 1; FPN, ferroportin; FSP1, ferroptosis suppressor protein 1, which requires the quinol cycle to attenuate lipid peroxidation; FTH1, ferritin heavy chain; FTL; ferritin light chain; GCLC, glutathione cysteine ligase catalytic subunit; GCLM, glutathione cysteine ligase modulator; GPx4, glutathione peroxidase 4; GSH, glutathione; GSSG, glutathione disulfide; GSTA4, glutathione S-transferase A4; HMOX; heme oxygenase; IRP, iron regulatory protein; LAT1, large neutral amino acid transporter 1; LOOH, Lipid hydroperoxides; Nrf2, Nuclear factor erythroid 2-related factor 2; Prdx6, peroxiredoxin 6; TF, transferrin, TfR; Transferrin receptor; xCT, cysteine-glutamate antiporter.

neuroscience↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2021

A native mass spectrometry-based assay for rapid assessment of the empty/full capsid ratio in AAV gene therapy products

Adeno-associated virus (AAV)-based cell and gene therapy is a rapidly developing field, requiring analytical methods for detailed product characterization. One important quality attribute of AAV products that requires monitoring is the amounts of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. Here, we present a native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation. We report rapid determination of the amount of empty particles in AAV5 and AAV8 samples, with results correlating well with more conventional analysis strategies, demonstrating the potential of state-of-the-art mass spectrometry for the characterization of viral particles.

biochemistry↗