Biomedical subjects
Paul S Freemont
Publications and source records attributed to Paul S Freemont.
Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model.
p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function.
Human PLU-1 Has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9.
PLU-1 is a large (1544 amino acids) nuclear protein that is highly expressed in breast cancers and is proposed to function as a regulator of gene expression. A yeast two-hybrid screen using PLU-1 as bait has identified two unrelated PLU-1 interacting proteins, namely brain factor-1 (BF-1) and paired box 9 (PAX9), both of which are developmental transcription factors. BF-1 and PAX9 interact with PLU-1 via a novel conserved sequence motif (Ala-X-Ala-Ala-X-Val-Pro-X4-Val-Pro-X8-Pro, termed the VP motif), because deletion or site-directed mutagenesis of this motif in either protein abolishes PLU-1 interaction in vivo. In a reporter assay system, PLU-1 has potent transcriptional repression activity. BF-1 and PAX9 also represses transcription in the same assay, but co-expression of PLU-1 with BF-1 or PAX9 significantly enhances this repression. Mutation of the PLU-1 binding motifs in BF-1 and PAX9 abolishes the observed PLU-1 co-repression activity. These data support a role for PLU-1 acting as a transcriptional co-repressor of two unrelated developmental transcription factors. Because both BF-1 and PAX proteins interact with members of the groucho co-repressor family, it is plausible that PLU-1 has a role in groucho-mediated transcriptional repression.
Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.
Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.
Machinery of protein folding and unfolding.
During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding. It has emerged that dual functionality in terms of folding and unfolding might exist for some systems. The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.