Biology subjects

Healy, M. D.

Publications and source records attributed to Healy, M. D..

7 recordsLinked to original sources

bioRxiv2026

Global identification of neuronal and astrocytic integral membrane proteins that require Retromer for their endosomal recycling.

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1225eedorg.highwire.dtl.DTLVardef@1863f1dorg.highwire.dtl.DTLVardef@99c21eorg.highwire.dtl.DTLVardef@976bfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

molecular biology↗

bioRxiv2026

A remarkable case of conserved domain swapping in the COMMD family of proteins

The eukaryotic COMMD family of proteins are core subunits of the Commander protein complex, with a central role in endosomal membrane trafficking and signalling. Previous crystal and cryoEM structures show that COMMD and COMMD-like proteins form homo-oligomeric and hetero-decameric assemblies with a central ring structure formed by their small C-terminal COMM domains. Their -helical N-terminal (HN) domains decorate each side of these rings, and in the eukaryotic Commander complex engage the coiled-coil proteins CCDC22 and CCDC93. Here, we have determined new crystal structures of the isolated HN domains of human Commd4, Commd9 and Commd10, and find that all three proteins form domain swapped structures with a remarkably consistent topology. This occurs via a conformational change in a hinge-loop between helices 2 and 3, leading to exchange of helices 3 to 6 between protomers. The hinge-loops of Commd9 and Commd10 possess several serine and threonine residues that can be phosphorylated, and we find that specific phospho-mimicking mutations in Commd10 can promote or inhibit domain swapping. Whether the unique COMMD HN domains play any roles beyond assembly with CCDC proteins is unclear, but this work suggests a common conformational switch exists with a potential to regulate their function.

biochemistry↗

bioRxiv2025

The prokaryotic origins of the COMMD protein family involved in eukaryotic membrane trafficking

The ten eukaryotic COMMD proteins are core components of the Commander complex, with central roles in endosomal membrane trafficking and signalling. Each protein has an -helical N-terminal (HN) domain with a C-terminal copper metabolism gene MURR1 (COMM) domain. These ten family members assemble into a heterodecameric ring composed of five specific heterodimers. In this work we have combined structural homology searches with genome-wide predicted structures to identify ancestral COMMD-like proteins that exist as single genes in Bacteria and Archaea. Although there is limited sequence similarity to the eukaryotic proteins the bacterial and archaeal COMMD-like proteins are predicted to form homomeric ring-shaped assemblies like their eukaryotic counterparts. Our biophysical studies, crystal and cryo-EM structures confirm COMMD-like proteins readily form homooligomeric rings composed of eight or ten subunits assembled from core dimeric building blocks and inter-dimer interactions that are analogous to the heterodecameric core structure of the eukaryotic Commander complex. Phylogenetic analyses using amino acid sequences and FoldSeek structural alphabet (3Di) infer that the closest identified relatives to the eukaryotic COMMD proteins are found in Myxococcota bacteria. These findings indicate that COMMD genes emerged early in eukaryotic evolution through multiple rounds of duplication from a single ancestral gene likely acquired from bacteria.

biochemistry↗

bioRxiv2025

Identification of a VPS29 isoform with restricted association to Retriever and Retromer accessory proteins through auto-inhibition.

The endosomal-lysosomal network is a hub of organelles that orchestrate the dynamic sorting of hundreds of integral membrane proteins to maintain cellular homeostasis. VPS29 is a central conductor of this network through its assembly into Retromer, Retriever and Commander endosomal sorting complexes, and its role in regulating RAB GTPase activity. Two VPS29 isoforms have been described, VPS29A and VPS29B, that differ solely in their amino-terminal sequences. Here we identify a third VPS29 isoform, which we term VPS29C, that harbours an extended amino-terminal sequence compared to VPS29A and VPS29B. Through a combination of AlphaFold predictive modelling, in vitro complex reconstitution, mass spectrometry and molecular cell biology, we find that the amino-terminal VPS29C extension constitutes an autoinhibitory sequence that limits access to a hydrophobic groove necessary for effector protein recruitment to Retromer, and association with Retriever and Commander. VPS29C is therefore unique in its ability to uncouple Retromer-dependent cargo sorting from the broader roles of VPS29A and VPS29B in regulating the endosomal-lysosomal network through accessory protein recruitment. Our identification and characterisation of VPS29C points to additional complexity in the differential subunit assembly of Retromer, an important consideration given the increasing interest in Retromer as a potential therapeutic target in neurodegenerative diseases. SIGNIFICANCE STATEMENTThe endosomal-lysosomal network is essential for normal cellular function with network defects being associated with numerous neurodegenerative diseases. Two heterotrimeric complexes, Retromer and Retriever, control transmembrane protein recycling through the network. Of these, reduced Retromer expression is observed in Alzheimers disease and Retromer mutations lead to familial Parkinsons disease. Here, we identify and characterise a new isoform of VPS29, a subunit shared between Retromer and Retriever. We reveal how this isoform, VPS29C, adopts an auto-inhibitory conformation to limit its association into Retriever and restrict the binding of VPS29C-containing Retromer to accessory proteins vital for regulating network function. By revealing added complexity in Retromer assembly and function, we provide new insight into Retromers potential as a therapeutic target in neurodegenerative diseases.

cell biology↗

bioRxiv2025

Identification of a RAB32-LRMDA-Commander membrane trafficking complex reveals the molecular mechanism of human oculocutaneous albinism type 7

Commander is an endosome associated sixteen protein assembly that associates with the sorting nexin 17 (SNX17) cargo adaptor to regulate cell surface recycling of internalised integral membrane proteins including integrins and lipoprotein receptors. Mutations in Commander are causative for Ritscher-Schinzel syndrome (RSS), a multiorgan developmental disease associated with a core triad of cerebellar-cardiac-craniofacial malformation. Here, using unbiased proteomics and computational modelling, we identify leucine rich melanocyte differentiation associated (LRMDA) as a novel Commander binding protein. Using recombinant protein reconstitution, we show that LRMDA simultaneously associates with Commander and active RAB32, and, by revealing that LRMDA and SNX17 share a common mechanism of Commander association, establish the mutually exclusive nature of RAB32-LRMDA-Commander and SNX17-Commander assemblies. From functional analysis in human melanocytes, we establish an essential role for RAB32-LRMDA-Commander in melanosome biogenesis and pigmentation and reveal a distinct functional role for SNX17-Commander in this organelle biogenesis pathway. We reveal how LRMDA mutations, causative for oculocutaneous albinism type 7 (OCA7), a hypopigmentation disorder accompanied by poor visual acuity, uncouple RAB32 and Commander binding thereby establishing the mechanistic basis of this disease. Our discovery and characterisation of this alternative Commander assembly establishes an unrecognised plasticity of Commander function within a highly complex organelle biogenesis pathway. This extends Commander function beyond the confines of SNX17-mediated cell surface recycling into RAB32-family mediated biogenesis of lysosome-related organelles and, potentially, other RAB32 regulated pathways including host-pathogen defence mechanisms. Our work also extends the breath of Commander pathway dysfunction for human disease.

cell biology↗

bioRxiv2024

Mechanism and regulation of cargo entry into the Commander recycling pathway

Commander is a multiprotein complex that orchestrates endosomal recycling of diverse integral cargo proteins and in humans is required for normal skeletal, brain, kidney, and cardiovascular development. While the structure of this complex has recently been described, the central question of how cargo proteins are selected for entry into the Commander recycling pathway remains unclear. Here using recombinant protein reconstitution and in silico predictions we identify the evolutionary conserved mechanism through which the unstructured carboxy-terminal tail of the integral protein adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the carboxy tail for Retriever association. Using molecular cell biology and high-resolution microscopy, we establish the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains for entry into the recycling pathway. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting complex.

cell biology↗

bioRxiv2021

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

biochemistry↗