Biology subjects

Hierro, A.

Publications and source records attributed to Hierro, A..

3 recordsLinked to original sources

bioRxiv2024

Retromer-mediated recruitment of the WASH complex involves discrete interactions between VPS35, VPS29 and Fam21

Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin- polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of Fam21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from Fam21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between Fam21 and retromer occurring near the Parkinsons disease-linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with Fam21 in vitro.

biochemistry↗

bioRxiv2023

Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo. Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

molecular biology↗

bioRxiv2023

Sequence independent activity of a predicted long disordered segment of the human papillomavirus L2 capsid protein during virus entry

The papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm during virus entry to bind cellular factors required for intracellular virus trafficking. Cytoplasmic protrusion of HPV16 L2, virus trafficking, and infectivity are inhibited by large deletions in an [~]110 amino acid segment of L2 that is predicted to be disordered. The activity of these mutants can be restored by inserting protein segments with diverse compositions and chemical properties into this region, including scrambled sequences, a tandem array of a short sequence, and the intrinsically disordered region of a cellular protein. The infectivity of mutants with small in-frame insertions and deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or its composition, determines its activity during virus entry. Sequence independent but length dependent activity has important implications for protein function and evolution.

biochemistry↗