Platelet-derived growth factor-B (PDGF-B) is important for normal tissue growth and maintenance and its overexpression has been linked to several diseases, including cancer, fibrotic disease and atherosclerosis. Here, we show that synthesized as a precursor, proPDGF-B is converted to a mature form by proteolytic cleavage at two sites and its N-terminal cleavage is a prerequisite for processing at its C-terminus. The first cleavage occurs at residues RGRR 81 k, and the second cleavage close to residues ARPVT 190 , just before the Cterminal amino-acid sequence crucial for PDGF-B retention to cell surface. Cotransfection of a Furin-deficient cell line LoVo-C5 with proPDGF-B and different PC members revealed that Furin, PACE4, PC5, and PC7 are candidate proPDGF-B convertases. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of proPDGF-B. The processing of proPDGF-B is blocked by site-directed mutagenesis of the RGRR 81 k sequence and by various PC inhibitors. Mutation of the PDGF-A and/or PDGF-B convertase sites, revealed that processing of both A and B chains is required for the formation of mature PDGF-B dimers and that the processing of the B chain controls the level of secreted and matrix-bound PDGF-BB forms. Our findings emphasize the importance of the convertasedirected processing of proPDGF-B at the RGRR 81 k sequence for PDGF-B maturation and secretion. Oncogene (2005) 24, 6925-6935.

There is growing appreciation that the nucleus is organized into an array of discrete structural domains, each subserving a specific function. These functional nuclear bodies are to be distinguished from pathological intranuclear inclusions which have been described in a variety of neurodegenerative diseases. Marinesco bodies (MBs) are eosinophilic ubiquitinated intranuclear inclusions found in pigmented neurons of the human substantia nigra and locus coeruleus. Traditionally considered non-pathological entities, more recent studies have indicated that MBs are associated with the age-associated degenerative changes in the substantia nigra and striatal loss of dopaminergic terminals. In the present morphological study of the human substantia nigra, we demonstrate colocalization, contiguity, and focally shared immunoreactivity between MBs and neuronal intranuclear rodlets (INRs). The latter nuclear structures of uncertain function are markedly decreased in the cortex of Alzheimer's disease, but not dementia with Lewy bodies. In addition, we demonstrate an interaction between INRs and promyelocytic leukemia (PML) protein, the signature protein of PML nuclear bodies. These results suggest that structures which subserve the functional compartmentalization of the neuronal nucleus may be relevant to elucidating cellular mechanisms of age-related motor dysfunction.

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In a previous study, we demonstrated immunoreactivity of a subset of neuronal intranuclear rodlets (INRs) in the human substantia nigra for promyelocytic leukaemia (PML) protein, the signature protein of PML bodies. In the present study, we extend these observations and describe the ultrastructural features, immunohistochemical staining characteristics, and topographical pattern of distribution of PML-immunoreactive intranuclear rodlets (PML-INRs). Consistent with a purported role for PML bodies in nuclear proteolysis and/or transcriptional regulation, PML-INRs are immunoreactive for components of the ubiquitin-proteasome system, the transcriptional regulator CREB-binding protein, acetylated histone H4, and the eukaryotic translation initiation factor eIF4E. Immunoelectron microscopy reveals that they all possess a filamentous core and, in some, this is surrounded by a granular shell. We further demonstrate that a proportion of INRs in extranigral sites also show partial immunoreactivity for PML. These observations indicate an intimate association between two neuronal nuclear bodies, PML bodies and INRs. Because both of these structures have been implicated in neurodegenerative disease, PML-INRs may provide a tool with which to study changes in nuclear substructure in disease.

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