Biology subjects

Hackney, C. M.

Publications and source records attributed to Hackney, C. M..

6 recordsLinked to original sources

bioRxiv2026

BAG6 and RNF126 are broadly involved in protein quality control of non-native missense protein variants

Protein quality control (PQC) degradation is a vigorous and selective system that limits the accumulation of harmful, non-native and aggregation-prone proteins. Specific PQC pathways have been described for misfolded ER proteins, aberrant translational products and mislocalized proteins. However, many components involved in PQC of structurally unstable cytosolic proteins are unknown despite such proteins being a common consequence of pathogenic coding variants. Here, we present results from a genome-wide CRISPR knockout screen to identify components involved in PQC of the Parkin R42P variant. We find that HSP90 and STIP1 stabilize Parkin, while the BAG6 chaperone and E3 ubiquitin ligase RNF126 are critical for PQC degradation. Variant abundance by massively parallel sequencing (VAMP-seq) reveal >1000 Parkin variants, including several variants linked to autosomal recessive juvenile Parkinsonism, are BAG6 targets. Pathogenic missense variants in phenylalanine hydroxylase (PAH) and the tumor suppressor FLCN are also stabilized in BAG6 and RNF126 knockout cells. We propose that BAG6 and RNF126 are broadly involved in PQC and may represent targets for the development of therapeutics for protein misfolding diseases.

biochemistry↗

bioRxiv2025

Blocking protein quality control degradation leads to structural stabilization of DHFR indel variants

Gene variants leading to insertions or deletions of amino acid residues (indels) often have detrimental consequences for the folding of the encoded protein. Yet at some positions indels are tolerated or result merely in partial unfolding and hypomorphic phenotypes. Typically unfolded proteins are targeted for protein quality control (PQC) degradation via the ubiquitin-proteasome system, which in yeast is mediated by specific E3 ubiquitin-protein ligases, including Ubr1 and San1. Here we systematically probed the folding of a library of indel variants in the DHFR protein using a sensitive yeast-based protein folding reporter. We show that deletion of Ubr1 or San1 leads to a greater fraction of folded DHFR indel variants, primarily positioned towards the N- and C-terminal regions in DHFR. Intriguingly, most of the DHFR indels that are structurally stabilized in the E3 knockout strains, are also stabilized at lowered temperatures and upon binding the DHFR inhibitor methotrexate. This suggests that blocking PQC degradation can restore function to partially unfolded hypomorph variants, thus providing a potential therapeutic avenue for protein misfolding diseases.

biochemistry↗

bioRxiv2025

Conkazal-M1 from the MKAVA family of conotoxins - a dual-function protease inhibitor and neuroactive peptide

Marine cone snails produce a diverse array of bioactive peptides, known as conotoxins, in their venom. Given their high target potency and specificity, conotoxins are attractive compounds for the development of precision research tools and pharmacological agents. Here, we provide the first experimental characterization of a conotoxin from the MKAVA superfamily, conkazal-M1, from Conus magus. Using NMR spectroscopy, we show that conkazal-M1 adopts a fold characteristic of the Kazal-type protease inhibitor family, featuring a Glu residue at the inhibitory P1 position. Recombinantly expressed conkazal-M1 inhibits the proteolytic activity of Subtilisin A with an apparent Ki of 1.1 M. In addition, conkazal-M1 partially inhibits calcium transients in mouse sensory neurons, suggesting a potential role in modulating ion-channel activity, as seen for many other toxins. The dual function of conkazal-M1 in protease inhibition and neuroactivity is analogous to the dual function of several toxins harboring a Kunitz-type fold. The well-conserved sequence of the MKAVAs indicates an evolutionary trajectory in which these proteins face an adaptive conflict, where mutations that enhance one activity compromise the other. Collectively, this work provides new structural and functional insights into a previously uncharacterized toxin superfamily in cone snails, illustrates how structural scaffolds can be repurposed for functions that diverge from the original while retaining their overall structure, and expands our understanding of the toxin arsenal available to venomous animals.

biochemistry↗

bioRxiv2025

Structural similarities reveal an expansive conotoxin family with a two-finger toxin fold

Venomous animals have evolved a diverse repertoire of toxins with considerable pharmaceutical potential. The rapid evolution of peptide toxins, such as the conotoxins produced by venomous marine cone snails, often complicates efforts to infer their evolutionary relationships based solely on sequence information. Structural bioinformatics, however, can provide robust support. Here, we first solve the NMR structure of a macro-conotoxin from the MLSML superfamily, Tx33.1, which is composed of 124 residues, including 12 cysteines. We then apply deep learning-based methods for structure prediction and comparison to identify structural similarities between this toxin and five additional, previously uncharacterized conotoxin superfamilies. Although only three of these superfamilies exhibit sequence homology, a combined approach incorporating structure prediction, structure comparison, and gene structure analysis supports the conclusion that all six superfamilies share a common evolutionary past. The Tx33.1 NMR structure displays similarity to the first two domains of Argos, a secretory protein from Drosophila melanogaster that comprises three domains, each harboring two short {beta}-stranded loops ("fingers"). Consequently, we propose the name "two-finger toxin (2FTX)" fold for this type of domain. Finally, using structure similarity searches, we identify a wide range of 2FTX proteins in protostomes, including non-venom-derived, secretory cone snail proteins. This study demonstrates how structural bioinformatics can be employed to uncover evolutionary relationships among rapidly evolving genes. It simultaneously identifies a large, previously unrecognized group of protostome 2FTX proteins, many of which exhibit close structural similarity to Argos and may perform a similar function in regulating EGFR signaling.

evolutionary biology↗

bioRxiv2022

Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and two additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.

biochemistry↗

bioRxiv2017

Intermolecular Disulfide-Bond Formation In Human FICD Modulates The Activity Of The Hyperactive E234G Mutant

Endoplasmic reticulum (ER) stress that leads to the accumulation of misfolded proteins in the ER initiates the unfolded protein response (UPR). This homeostatic response activates signaling pathways that seek to reinstate a proper ER protein folding balance or induce apoptosis if ER stress persists. Recently, we and others identified human FICD (Filamentation induced by cyclic AMP domain-containing protein), an enzyme with adenylyltransferase (aka AMPylation) activity, as a new UPR target. Here, we demonstrate that FICD is functionally linked to the UPR, as evidenced by the finding that the adenylyltransferase activity of the protein induces ER stress, while FICD silencing increases sensitivity to ER stress. We identify BiP, an abundant ER chaperone and key regulator of the UPR, as the main substrate of FICD AMPylation in ER-derived microsomes, further emphasizing close functional connection of FICD to the UPR and in line with recent reports that AMPylation inactivates BiP. Notably, BiP overexpression increased the levels of BiP AMPylation as well as FICD auto-AMPylation, suggesting a homeostatic response that balances the pool of active BiP to modulate its functions in protein folding as well as UPR signaling. Finally, we show that overexpressed FICD forms a disulfide-bonded homo-dimer through Cys51 and Cys75 and demonstrate that mutation of these two cysteines in the context of a hyperactive FICD mutant leads to increased BiP AMPylation. This latter finding opens up the possibility that FICD activity is redox regulated and closely connected with ER redox homeostasis.

molecular biology↗