👤 Marja M Hurley

Sickle cell disease (SCD) is characterized by osteopenia and impaired bone mineralization, but the underlying mechanisms remain unclear. Fibroblast growth factor 23 (FGF23), elevated in SCD, regulates phosphate metabolism through FGFRs/klotho and contributes to bone loss. Although FGF23's systemic effects are known, its local actions in SCD bone remain poorly defined. Using bone marrow stromal cells (BMSCs) derived from SCD mice, we previously reported that enhanced local FGF23/FGFR1 signaling and increased osteopontin impair osteoblast mineralization, which is rescued by an FGF23-neutralizing antibody (FGF23Ab). Here, we further investigated downstream signaling and pyrophosphate/phosphate (PPi/Pi)-regulatory mechanisms contributing to mineralization defects. FGF23Ab reduced phospho-FGFR1, restored phospho-FGFR2 and phospho-AKT, and decreased pSTAT3 activation. SCD-BMSCs exhibited increased matrix inhibitors, matrix Gla protein (MGP) and matrix extracellular phosphoglycoprotein (MEPE), and reduced mineralization promoters PHEX and DMP1, which were partially normalized by FGF23Ab. FGF23Ab also corrected elevated PPi-generating enzymes ENPP1 and ANK and restored tissue-nonspecific alkaline phosphatase (TNAP). In contrast, the phosphate importer PiT2 was significantly reduced in SCD BMSCs and was further suppressed with FGF23Ab. These findings indicate that excessive local FGF23 signaling disrupts mineralization by upregulating matrix inhibitors and altering PPi/Pi-regulatory pathways. FGF23 neutralization partially restores mineralization capacity. Show less

The ESCRT-0 and ESCRT-I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85-Å crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centred on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP-binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is non-essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms. Show less

The ESCRT complexes are required for multivesicular body biogenesis, macroautophagy, cytokinesis, and the budding of HIV-1. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA+ ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-III substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the betadomains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the two-fold axis of the Vta1 VSL domain is parallel to within approximately 20 degrees of the six-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly. Show less

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 A resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole. Show less