Biology subjects

Wood, K. M.

Publications and source records attributed to Wood, K. M..

4 recordsLinked to original sources

bioRxiv2026

Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

The obligate intracellular tick-borne pathogen Anaplasma phagocytophilum is unusual in its tropism for neutrophils, within which it survives and replicates. Central to the ability of this bacterium to colonize this hostile niche are secreted effector proteins. One such effector protein, AnkA, interacts with multiple host ligands in both the cytoplasm and the nucleus, yet how these interactions promote infection is largely unknown. We discovered that AnkA directly binds both the WAVE Regulatory Complex (WRC), a signal-integration hub that regulates the formation of branched actin networks underpinning diverse cell functions, and to monomeric and filamentous actin. We determined that AnkA uses two forms of molecular mimicry, first by binding to the Abi1 component of the WRC via a WRC-Recruiting Abi-binding Peptide (WRAP), and second by binding to actin via a WH2-like Actin Binding Motif (ABM). Via these interactions, AnkA can exert multi-factorial influence on the actin cytoskeleton and signaling pathways converging on this host structure. AnkA and the WRC co-localise during bacterial internalization and during the late stages of infection, suggesting a central role in both host-cell colonization and bacterial dissemination. Our work reveals AnkA as the first example of a bacterial effector that directly targets the WRC. The co-occurrence of WRAP- and ABM-like motifs in other bacterial effector proteins suggests that this mechanism of actin cytoskeleton subversion may represent a conserved strategy used by other obligate intracellular bacteria.

microbiology↗

bioRxiv2021

Multi-modal adaptor-clathrin contacts drive coated vesicleassembly

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the {beta}2 subunit of AP2, and several binding sites on {beta}2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of {beta}2 hinge and appendage ({beta}2HA) domains. We find that the {beta}2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, {beta}2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of {beta}2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage, with the appendage "platform site" having less importance. From these studies and the work of others, we propose that {beta}2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

biochemistry↗

bioRxiv2020

Defining Substrate Requirements for Cleavage of Farnesylated Prelamin A by the Integral Membrane Zinc Metalloprotease ZMPSTE24

The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24s selectivity for prelamin A.

cell biology↗

bioRxiv2019

Discovery of conformation-sensitive anti-amyloid protofibril monoclonal antibodies using an engineered chaperone-like amyloid-binding protein

The hypothesis that amyloid beta peptides (A{beta}) are central to the pathogenesis of sporadic Alzheimers disease (AD) is still hotly debated. Although several monoclonal antibodies (mAbs) against A{beta} have failed in therapeutic clinical trials, two conformation-selective, anti-A{beta} mAbs continue to show promise. A significant challenge has been to discover mAbs that preferentially target A{beta} protofibrils over natively-folded monomeric peptides or amyloid plaques. We have engineered a novel chaperone-like amyloid-binding protein (CLABP), Nucleobindin 1 (NUCB1), which enables the stabilization of protofibrils, allowing them to be used as immunogens in mice to facilitate the generation of mAbs that recognize A{beta} protofibrils. An immunization campaign and subsequent screening funnel identified a panel of mAbs with high-affinity to A{beta}. Two mAbs in particular, 1A8 and 7C8, displayed significant conformation sensitivity and preferentially bound A{beta} protofibrils over monomers. Furthermore, 1A8 delayed A{beta} aggregation, but did not prevent eventual fibril formation, while 7C8 significantly and dose-dependently reduced fibril formation by inhibiting both primary and secondary nucleation. Both mAbs protected against protofibril-induced cytotoxicity in vitro, and showed distinctive staining patterns by immunohistochemistry in PS1/APP mice and in post-mortem AD brain tissue. In summary, we describe a novel method to stabilize soluble A{beta} protofibrils for use in immunization campaigns. We hypothesize that the stabilized protofibrils retain the neoepitopes of the A{beta} protofibril and the aggregates found in mouse models of disease and post-mortem AD brain tissue.

molecular biology↗