Shuiliang Yu

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrPC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

A multistage pathway for human prion protein aggregation in vitro: from multimeric seeds to β-oligomers and nonfibrillar structures.

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.

Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.

The stability and aggregation of ovine prion protein associated with classical and atypical scrapie correlates with the ease of unwinding of helix-2.

Susceptibility to scrapie disease in sheep, the archetypal prion disease, correlates with polymorphisms within the ovine PrP (prion-related protein) gene. The VRQ (Val136Arg154Gln171) and AL141RQ (Ala136Leu141Arg154Gln171) allelic variants are associated with classical scrapie, whereas the ARR (Ala136Arg154Arg171), AF141RQ (Ala136Phe141Arg154Gln171) and AHQ (Ala136His154Gln171) allelic variants are associated with atypical scrapie. Recent studies have suggested that there are differences in the stability of PrPSc (abnormal disease-specific conformation of PrP) associated with these different forms of scrapie. To address which structural features of ovine PrP may contribute to this difference, in the present study we have investigated the conformational stability and susceptibility to aggregation of allelic variants of ovine PrP associated with classical or atypical scrapie. We find that the melting temperature of ovine recombinant VRQ and AL141RQ PrP is higher than that of AF141RQ, AHQ and ARR. In addition, monoclonal-antibody studies show that the region around helix-1 of VRQ and AL141RQ is less accessible compared with other ovine PrP allelic variants. Furthermore, the extent of both the structural change to copper-ion-treatment and denaturant-induced aggregation was reduced in PrP associated with atypical scrapie compared with PrP associated with classical scrapie. Through the use of molecular dynamics simulations we have found that these biochemical and biophysical properties of ovine PrP correlate with the ease of unwinding of helix-2 and a concurrent conformational change of the helix-2-helix-3 loop. These results reveal significant differences in the overall stability and potential for aggregation of different allelic variants of ovine PrP and consequently have implications for the differences in stability of PrPSc associated with classical and atypical scrapie.

Ligand binding promotes prion protein aggregation – role of the octapeptide repeats

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu2+ and Zn2+. Here, we report our findings that GAG and Cu2+ promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu2+, mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild‐type PrP. When the GAG‐binding motif, KKRPK, is deleted the effect of GAG but not that of Cu2+ is abolished. By contrast, when the Cu2+‐binding motif, the octapeptide‐repeat region, is deleted, neither GAG nor Cu2+ is able to promote aggregation. Therefore, the octapeptide‐repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti‐PrP mAbs, whereas none of the tested anti‐PrP mAbs block Cu2+‐promoted aggregation. However, a mAb that is specific for an epitope at the N‐terminus enhances aggregation in the presence of either GAG or Cu2+. Therefore, although binding of either GAG or Cu2+ promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.

Aggregation of prion protein with insertion mutations is proportional to the number of inserts

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

Binding of recombinant but not endogenous prion protein to DNA causes DNA internalization and expression in mammalian cells.

Recombinant prion protein, rPrP, binds DNA. Both the KKRPK motif and the octapeptide repeat region of rPrP are essential for maximal binding. rPrP with pathogenic insertional mutations binds more DNA than wild-type rPrP. DNA promotes the aggregation of rPrP and protects its N terminus from proteinase K digestion. When rPrP is mixed with an expression plasmid and Ca2+, the rPrP·DNA complex is taken up by mammalian cells leading to gene expression. In the presence of Ca2+, rPrP by itself is also taken up by cells in a temperature- and pinocytosis-dependent manner. Cells do not take up rPrPΔKKRPK, which lacks the KKRPK motif. Thus, rPrP is the carrier for DNA and the KKRPK motif is essential for its uptake. When mixed with DNA, a pentapeptide KKRPK, but not KKKKK, is sufficient for DNA internalization and expression. In contrast, whereas the normal cellular prion protein, PrPC, on the cell surface can also internalize DNA, the imported DNA is not expressed. These findings may have relevance to the normal functions of PrPC and the pathogenic mechanisms of human prion disease.

Notch Receptor‐Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention

Notch is long recognized as a signaling molecule important for stem cell self‐renewal and fate determination. Here, we reveal a novel adhesive role of Notch‐ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O‐fucosylglycans on Notch EGF‐like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand‐expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand‐bearing osteoblastic or stromal cells inhibits wild type but not O‐fucosylglycan‐deficient HSPC cycling, independent of RBP‐JK‐mediated canonical Notch signaling. Furthermore, Notch‐ligand neutralizing antibodies induce RBP‐JK‐independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor–ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O‐fucosylglycans on Notch. Stem Cells 2015;33:2280–2293

Aberrant Notch Signaling in the Bone Marrow Microenvironment of Acute Lymphoid Leukemia Suppresses Osteoblast-Mediated Support of Hematopoietic Niche Function

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.