Sequence variation in the 3'-untranslated region of the dopamine transporter gene and attention-deficit hyperactivity disorder (ADHD).

The dopamine transporter gene (DAT1) has been reported to be associated with attention-deficit hyperactivity disorder (ADHD) in a number of studies [Cook et al. (1995): Am J Human Genet 56(4):9993-998; Gill et al. (1997): Mol Psychiatry 2(4):311-313; Waldman et al. (1998): Am J Human Genet 63(6):1767-1776; Barr et al. (2001): Biol Psychiatry 49(4):333-339; Curran et al. (2001): Mol Psychiatry 6(4):425-428; Chen et al. (2003): Mol Psychiatry 8(4):393-396]. Specifically, the 10-repeat allele of the 40-bp variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region (UTR) of the gene has been found to be associated with ADHD. There is evidence from in vitro studies indicating that variability in the repeat number, and sequence variation in the 3'-UTR of the DAT1 gene may influence the level of the dopamine transporter protein [Fuke et al. (2001): Pharmacogenomics J 1(2):152-156; Miller and Madras (2002): Mol Psychiatry 7(1):44-55]. In this study, we investigated whether DNA variation in the DAT1 3'UTR contributed to ADHD by genotyping DNA variants around the VNTR region in a sample of 178 ADHD families. These included a MspI polymorphism (rs27072), a DraI DNA change (T/C) reported to influence DAT1 expression levels, and a BstUI polymorphism (rs3863145) in addition to the VNTR. We also screened the VNTR region by direct resequencing to determine if there was sequence variation within the repeat units that could account for the association. Our results indicate that DAT1 is associated with ADHD in our sample but not with alleles of the VNTR polymorphism. We did not find any variation in the sequence for either the 10- or 9-repeat alleles in the probands screened nor did we observe the reported DraI (T/C) variation. Our results therefore refute the possibility of the reported DraI variation or alleles of the VNTR as the functional variants contributing to the disorder.     

Clinical and Genetic Studies of Familial Hemophagocytic Lymphohistiocytosis in Oman: Need for Early Treatment

Hemophagocytic lymphohistiocytosis (HLH) embraces the frequently indistinguishable conditions of familial hemophagocytic lymphohistiocytosis (FHL) and virus-associated hemophagocytic syndrome (VAHS). Without therapy FHL is invariably fatal, but successful therapy, including chemotherapy and immunotherapy followed by bone marrow transplantation (BMT), has been presented. To clarify the outcome of HLH in a developing country, with regard to clinical, laboratory, and genetic features, a nationwide study on all patients diagnosed with HLH in Oman during the 5-year period 1997-2001 was performed. In 5 patients and their families, mutational analysis was made. Thirteen patients with HLH were identified, 5 of whom had clinical manifestations of central nervous system involvement at presentation. In none of the patients could an infectious cause be identified. Ten children were referred late in the disease course, and the concern about starting chemotherapy before exclusion of an acute viral infection resulted in delayed treatment in some patients. Two children were started early on the HLH-94-therapy followed by successful BMT in one child. In the successfully transplanted child, the response to intrathecal hydrocortisone appeared to be better than standard therapy with intrathecal methotrexate. Finally, a novel missense mutation in the perforin gene was identified in 2 patients and their family members, causing a transition of proline to threonine at codon 89. Early diagnosis and treatment is important to improve outcome. Intrathecal corticosteroids may be considered, in addition to intrathecal methotrexate, in certain patients. Since the novel perforin mutation has been reported in only 2 patients from Oman, and since similar polymorphism in the sequencing data of the members of their families has been identified, a founder effect is possible in this population.     

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1?? resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.     

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1–Pad1–N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.In eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1–5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6–8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM–based approaches (11, 12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independently (12, 14). The base contains the hetero-hexameric AAA-ATPase ring (Rpt1–Rpt6), which drives the conformational changes required for substrate processing, including unfolding and translocation into the CP (15, 16). The base also contains the largest RP non-ATPase subunits, Rpn1 and Rpn2, and the Ub receptor Rpn13. The second resident Ub receptor, Rpn10, is not part of either the base or the lid; it binds only to the assembled 26S proteasome and is positioned close to the ATPase module.The lid scaffold is composed of the Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, and Rpn12 subunits (14). These subunits can be grouped according to their domain structures. Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 each comprise an N-terminal helix repeat segment, a proteasome-COP9/signalosome-eIF3 (PCI) module, and a long helix at the C terminus (8). The Rpn8 and Rpn11 subunits each consist of an Mpr1–Pad1–N-terminal (MPN) domain, followed by long C-terminal helices (Fig. 1A). The PCI subunits form a horseshoe-shaped structure and the MPN domains form a heterodimer, which are connected by a large helical bundle, to which all subunits contribute (13, 17, 18). Each of these eight subunits has paralogs in the COP9/signalosome (CSN) and the elongation initiation factor 3 (eIF3), which likely adopt a similar architecture (18–21).Open in a separate windowFig. 1.Biochemical activity of the Rpn8-Rpn11 fusion protein. (A) Domain structures of Rpn8, Rpn11 and the fusion protein. (B) Ub₄ cleavage activity of 26S proteasome, WT Rpn8-Rpn11 and Rpn8-Rpn11 (E48Q). Cleavage of labeled peptide from Ub₄ was detected by the change in fluorescence polarization after 1hr incubation at 37 °C at the indicated concentrations. Values are normalized to maximum cleavage activity of 26S proteasome. The used 26S proteasome preparation contained only trace amounts of the DUB Ubp6.The lid strengthens the interaction between the CP and RP (17) and deubiquitylates substrates before their processing by the AAA-ATPase module and the CP. Cleavage of polyubiquitin chains from the substrate enables recycling of Ub into the cellular pool, and the removal of the unfolding-resistant Ub moieties promotes translocation of substrates. The MPN domain of Rpn11 contains the catalytic site for deubiquitylation (22, 23). Rpn11 belongs to the JAB1/MPN/Mov34 metalloenzyme (JAMM) family of metalloproteases, which provide the isopeptidase activities in the proteasome, CSN, and exo-deubiquitylating enzymes (DUBs), such as associated molecule with the SH3 domain of STAM-like protein (AMSH-LP). The signature motif for this family is a conserved glutamate upstream of a zinc-coordinating catalytic loop, H(S/T)HX₇SXXD, first revealed in the structure of an archaeal homolog, AfJAMM (24). The substrate-binding mode of JAMM DUBs was clarified by the crystal structure of AMSH-LP in complex with Lys63-linked diubiquitin (25). The other proteasomal MPN subunit, Rpn8, is catalytically inactive; it does not contain the JAMM motif and appears to have mainly a supporting role for Rpn11. Isolated Rpn11 is catalytically inactive, as is the isolated lid (22). Rpn11 is activated upon integration into the 26S holocomplex and is dependent on ATP hydrolysis (23). The 26S proteasome was recently shown to undergo large-scale conformational changes from a substrate-accepting conformation to a substrate-engaged conformation that may be critical for Rpn11 function (15, 26), but the mechanistic basis for the regulation of Rpn11 remains unclear. Loss-of-function mutants of the JAMM motif cause stalling of substrates above the mouth of the ATPase module and lead to clogging of the 26S proteasome (23, 26).Inhibitors of human Rpn11 (hRpn11, also known as POH1) have been proposed as potential antitumor agents working upstream of the β5 proteolytic subunits in the UPS. The β5 subunits have been clinically validated by the approval of bortezomib and carilfzomib for the treatment of hematologic malignancies. siRNA and mutagenesis studies show that expression of the zinc catalytic domain of hRpn11 is essential for cell survival (27). Inhibition of hRpn11 in combination with EGFR inhibition has been suggested to be beneficial in the treatment of nonsmall cell lung cancer (28). Overexpression of hRpn11 in cancer cells has been linked to their tumor escape from cytotoxic agents (29). Thus, hRpn11 is an attractive target for pharmacologic intervention of the UPS.Here we present three crystal structures of the catalytically active Rpn8/Rpn11 MPN heterodimer from Saccharomyces cerevisiae, revealing the details of the Rpn11 active site and the mode of interaction with other subunits. Not all structures show proper active site geometry, hinting at possible mechanisms preventing activation outside of the proteasome complex. The access path for the C-terminal peptide of the substrate-bound Ub is blocked by a highly conserved insertion specific to Rpn11. Fitting of the Rpn8-Rpn11 crystal structure into the cryo-EM density of both the substrate-accepting and substrate-engaged proteasome revealed how the subcomplex is situated between base and PCI domain subunits, which involves long insertions unique to Rpn11 and Rpn8. Contacts to the coiled coils and the oligosaccharide-binding fold (OB) domain ring of the AAA subunits appear to control active site geometry and proper access of the isopeptide bond segment. In the substrate-engaged proteasome, the catalytic center becomes situated just above the maw of the ATPase ring.