Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface. (Blood. 2014;124(8):1335-1343

BackgroundThe TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease.Principal FindingsWe analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10.ConclusionsTRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally.

The minichromosome maintenance (Mcm) 2-7 complex is the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G 1 , the Mcm2-7 complex is loaded onto chromatin to form prereplicative complex in a Cdt1-dependent manner. However, the detailed molecular mechanism of this loading process is still elusive. In this study, we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdt1 binding domain (CBD) and present its high resolution NMR structure. The structure of CBD exhibits a typical "winged helix" fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies, indicating that it is specific for protein-protein interaction. The CBDCdt1 interaction involves the helix-turn-helix motif of CBD. The results reported here provide insight into the molecular mechanism of Mcm2-7 chromatin loading and prereplicative complex assembly.For the maintenance of genetic integrity, initiation of eukaryotic DNA replication is tightly controlled to ensure that DNA replication occurs exactly once in each cell cycle. Replication begins by the formation of pre-RCs 4 on replication origins during late M and G 1 phases (1, 2). For pre-RC assembly, the sixsubunit origin recognition complex first binds replication origin on newly synthesized chromatin. The origin recognition complex serves as an origin marker and recruits the initiation factors Noc3p, Cdc6, and Cdt1 to origins for the chromatin loading of the heterohexameric Mcm2-7 complex (3-5). Once the Mcm complex is loaded onto chromatin and pre-RC is formed, the cell is licensed for DNA replication, awaiting additional signals for the activation of the licensed origins (6). The Mcm2-7 complex was first identified as a set of genes required for minichromosome maintenance in budding yeast ( Cdt1 is a critical member of pre-RC, and its main function is to load Mcm2-7 helicase onto chromatin to license the DNA for replication in the subsequent S phase (16). Overexpression of Cdt1 alone in many types of mammalian cells is sufficient to induce rereplication (17-19). Previous studies have broadly defined three functional domains of Cdt1: a domain in the middle of the molecule containing the major Geminin interaction site; an N-terminal domain, which is required for ubiquitin-mediated proteolysis and contains a second interaction site for Geminin; and a C-terminal domain, which is required for association with Mcm proteins (12). Interactions between Cdt1 and individual members of the Mcm2-7 complex have been examined, and Cdt1 was found to interact with Mcm2 and Mcm6 (16,[21][22][23]. The existence of a stoichiometric complex between Cdt1 and Mcm2-7 was recently reported, which is consistent with earlier biochemical and genetic investigations (24). However, the detailed molecular mechanism underlying the chromatin loading of the Mcm2-7 complex through Cdt1 remains elusive.In this report, w...

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