Functional analysis of novel mutations in y(+)LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI)

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals. LPI is caused by mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding y(+)LAT-1 (y(+)L amino acid transporter-1), which co-induces together with 4F2 heavy chain (4F2hc) system y(+)L in Xenopus oocytes. All Finnish LPI patients share the same founder mutation 1181-2A-->T (LPI(Fin)) not found in LPI patients elsewhere. Mutation screening of 20 non-Finnish LPI patients revealed 10 novel mutations: four deletions, two missense mutations, two nonsense mutations, a splice site mutation and a tandem duplication. Five LPI mutations (L334R, G54V, 1291delCTTT, 1548delC and LPI(Fin)) were studied functionally. All mutant proteins failed to co-induce amino acid transport activity when expressed with 4F2hc in Xenopus oocytes. Immunostaining experiments revealed that frameshift mutants 1291delCTTT, 1548delC and LPI(Fin)remained intracellular on expression with 4F2hc. In contrast, the missense mutants L334R and G54V reached the oocyte plasma membrane when co-expressed with 4F2hc, demonstrating that they are transport-inactivating mutations. This finding, together with the strong degree of conservation among all members of this family of amino acid transporters, indicates that residues L334 and G54 play a crucial role in the function of the y(+)LAT-1 transporter.     

Cationic amino acid transport through system y+L in erythrocytes of patients with lysinuric protein intolerance

We test the hypothesis that lysinuric protein intolerance (LPI), a rare autosomal recessive defect of cationic amino acid transport, results from the absence of the recently described y+L amino acid transporter. We compare fluxes of lysine (1 microM) into erythrocytes of normal subjects with those of patients homozygous for the LPI mutation. No significant differences in fluxes through system y+L in normal or LPI cells were found, excluding the possibility that system y+L cannot be expressed in patients with LPI. Reasons for supposing that there may be tissue-specific processing of two recently described genes encoding the y+L transporter are discussed. Polymerase chain reaction measurement of expression of these two genes in an erythroleukemic cell line suggests that alternatively there may be an as-yet-unidentified additional member of this gene family.     

Five novel SLC7A7 variants and y+L gene-expression pattern in cultured lymphoblasts from Japanese patients with lysinuric protein intolerance

Two distinct human light subunits of the heteromeric amino acid transporter, y+LAT-1 coded by SLC7A7 and y+LAT-2 coded by SLC7A6, are both known to induce transport system y+L activity. SLC7A7 has already been identified as the gene responsible for lysinuric protein intolerance (LPI). We successfully identified five novel SLC7A7 variants (S238F, S489P, 1630delC, 1673delG, and IVS3-IVS5del9.7kb) in Japanese patients with LPI by PCR amplification and direct DNA sequencing. In addition, we performed a semi-quantitative expression analysis of SLC7A7 and SLC7A6 in human tissue. In normal tissue, the gene-expression ratio of SLC7A6 to SLC7A7 was high in the brain, muscle, and cultured skin fibroblasts; low in the kidneys and small intestine; and at an intermediate level in peripheral blood leukocytes, the lungs, and cultured lymphoblasts. The gene-expression ratio of SLC7A6 to SLC7A7 in cultured lymphoblasts was significantly different between normal subjects and LPI patients with R410X and/or S238F, where the relative amount of SLC7A7 mRNA was significantly lower and the relative amount of SLC7A6 mRNA was statistically higher in affected lymphoblasts than in normal cells. Expression of SLC7A7 and SLC7A6 may thus be interrelated in cultured lymphoblasts.     

The first korean case of lysinuric protein intolerance: presented with short stature and increased somnolence

Lysinuric protein intolerance (LPI) is a rare inherited metabolic disease, caused by defective transport of dibasic amino acids. Failure to thrive, hepatosplenomegaly, hematological abnormalities, and hyperammonemic crisis are major clinical features. However, there has been no reported Korean patient with LPI as of yet. We recently encountered a 3.7-yr-old Korean girl with LPI and the diagnosis was confirmed by amino acid analyses and the SLC7A7 gene analysis. Her initial chief complaint was short stature below the 3rd percentile and increased somnolence for several months. Hepatosplenomegaly was noted, as were anemia, leukopenia, elevated levels of ferritin and lactate dehydrogenase, and hyperammonemia. Lysine, arginine, and ornithine levels were low in plasma and high in urine. The patient was a homozygote with a splicing site mutation of IVS4+1G > A in the SLC7A7. With the implementation of a low protein diet, sodium benzoate, citrulline and L-carnitine supplementation, anemia, hyperferritinemia, and hyperammonemia were improved, and normal growth velocity was observed.     

Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis

Both homozygous (L166P, M26I, deletion) and heterozygous mutations (D149A, A104T) in the DJ-1 gene have been identified in Parkinson's disease (PD) patients. The biochemical function and subcellular localization of DJ-1 protein have not been clarified. To date the localization of DJ-1 protein has largely been described in studies over-expressing tagged DJ-1 protein in vitro. It is not known whether the subcellular localization of over-expressed DJ-1 protein is identical to that of endogenously expressed DJ-1 protein both in vitro and in vivo. To clarify the subcellular localization and function of DJ-1, we generated three highly specific antibodies to DJ-1 protein and investigated the subcellular localization of endogenous DJ-1 protein in both mouse brain tissues and human neuroblastoma cells. We have found that DJ-1 is widely distributed and is highly expressed in the brain. By cell fractionation and immunogold electron microscopy, we have identified an endogenous pool of DJ-1 in mitochondrial matrix and inter-membrane space. To further investigate whether pathogenic mutations might prevent the distribution of DJ-1 to mitochondria, we generated human neuroblastoma cells stably transfected with wild-type (WT) or mutant (M26I, L166P, A104T, D149A) DJ-1 and performed mitochondrial fractionation and confocal co-localization imaging studies. When compared with WT and other mutants, L166P mutant exhibits largely reduced protein level. However, the pathogenic mutations do not alter the distribution of DJ-1 to mitochondria. Thus, DJ-1 is an integral mitochondrial protein that may have important functions in regulating mitochondrial physiology. Our findings of DJ-1's mitochondrial localization may have important implications for understanding the pathogenesis of PD.     

Male intolerance of depression: A review with implications for psychotherapy

Evidence for sex differences in the experience and expression of depression and in help-seeking behavior and depression coping responses is reviewed. The data support the proposition that depression for many men may be a private experience, unshared with others and possibly also hidden from others, that men attempt to alleviate or remove by their own efforts without external help. The thesis is presented with supporting evidence that men are intolerant of depression because the experience of depression is incompatible with the male sex role and masculine socialization. Evidence is also examined for the punishment of male expression of depression. Consequences of male intolerance of depression and, implications and recommendations for psychotherapy are discussed.     

CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.     

Epithelial membrane protein 2 (EMP2): A systematic review of its implications in pathogenesis

ObjectivesEpithelial membrane protein 2 (EMP2) is a cell surface protein composed of approximately 160 amino acids and encoded by the growth arrest-specific 3 (GAS3)/peripheral myelin protein 22 kDa (PMP22) gene family. Although EMP2 expression has been investigated in several diseases, much remains unknown regarding its mechanism of action and the extent of its role in pathogenesis. Our aim was to perform a systematic review on the involvement of EMP2 in disease processes and the current usage of anti-EMP2 therapies.MethodsA Boolean search of the English-language medical literature was performed. PubMed, Scopus, Cochrane, and Web of Science were used to identify relevant citations. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Results52 studies met the inclusion criteria for qualitative analysis. Of those, 28 (53.8%) were human-only studies, 11 (21.2%) were animal-only studies, and 13 (25%) studies included both human and animal models. Furthermore, 34 (65.4%) studies focused on EMP2’s role in neoplasms, while the remaining 18 (34.6%) articles evaluated its role in other pathologies.ConclusionOverall, the evidence suggests the mechanisms of action of EMP2 are context dependent. Promising results have been produced by utilizing EMP2 as a biomarker and therapeutic target. More studies are warranted to better understand the mechanism and comprehend the role of EMP2 in the pathogenesis of diseases.     

Immunomodulatory effects of the HIV-1 gp120 protein on antigen presenting cells: implications for AIDS pathogenesis

Antigen presenting cell (APC) function is central to the development of an effective anti-viral immune response. Among APC, monocytes, macrophages and dendritic cells (DC) form the principal non-T cell compartment involved in in vivo HIV infection, and these cells play important and well-established roles in multiple aspects of viral pathogenesis. HIV infection may result in APC defects, which could ultimately contribute to the loss of CD4+ T cell responses observed early in HIV infection, when the CD4+ T cell number is still within the normal range. Extensive in vitro studies have demonstrated that the envelope glycoproteins of HIV-1 exert profound influences on various cell populations of the immune system, including hematopoietic progenitors, T and B lymphocytes, monocytes/ macrophages and DC, as well as on neuronal cells. The demonstration of the presence of envelope proteins both free in the circulation and bound to the surface of CD4+ cells suggests that gp120 interactions with non-infected cells can influence cellular functions in vivo, thus contributing to the immunopathogenesis of AIDS. This paper provides an overview of the present knowledge on gp120 binding, signal transduction triggering and interference with macrophage and DC functions and it highlights the importance of this interaction in the pathogenesis of AIDS.     

A deficiency of cysteine impairs fibrillin-1 deposition: implications for the pathogenesis of cystathionine beta-synthase deficiency

Cystathionine beta-synthase (CBS) deficiency is an inborn error of amino acid metabolism that has pleiotropic manifestations and is commonly called "homocystinuria." The features include skeletal, ocular, and vascular defects, some of which are reminiscent of those found in Marfan syndrome (MFS). Because of the spectrum of clinical effects, the pathogenesis of homocystinuria has long been thought to involve the extracellular matrix (ECM), and the condition has been classified as a heritable disorder of connective tissue. Because of the superficial similarities with MFS, we and others (Pyeritz, in McKusicks Heritable Disorders of Connective Tissue, St. Louis, Mosby-Year Book Inc., 5th ed., pp 137-178, 1993; Pyeritz, in Principles and Practice of Medical Genetics, New York, Churchill Livingstone, 3rd ed., pp 1027-1066, 1997; Mudd, Levy, and Skovby, in The Metabolic and Molecular Bases of Inherited Disease, New York, McGraw-Hill Publishing Co., 7th ed., pp 1279-1327, 1995) have speculated how CBS deficiency might affect fibrillin-1, the protein altered in MFS. For example, the altered plasma concentrations of homocysteine and/or cysteine in patients with CBS deficiency may hinder fibrillin-1 synthesis, deposition, or both. When arterial smooth muscle cells were cultured under conditions of cysteine deficiency, fibrillin-1 deposition into the ECM was greatly diminished as revealed by immunocytochemistry. Excessive homocysteine, in contrast, had little, if any, effect on fibrillin-1 deposition. When cysteine concentrations were returned to normal, the smooth muscle cells began to accumulate a matrix rich in fibrillin-1. Type I collagen, the major matrix component synthesized by these smooth muscle cells, was not reduced by low cysteine concentrations nor high homocysteine concentrations. These results demonstrate that a deficiency of cysteine and subsequent inhibition of fibrillin-1 accumulation in CBS deficient patients may be at least partly responsible for their phenotype, and suggest that maintenance of normal plasma cyst(e)ine levels may be an important therapeutic goal.     

Identification of novel USH2A mutations: implications for the structure of USH2A protein

Usher syndrome type II is an autosomal recessive disorder, characterised by stable hearing impairment from childhood and progressive retinitis pigmentosa from the late teens. Mutations in the USH2A gene, located on 1q41, were recently shown to be responsible for Usher syndrome type IIa. We have investigated the molecular pathology of Usher type II by screening the USH2A gene for mutations in 31 unrelated patients from Denmark and Norway. Besides the frequent 2299delG mutation, which accounted for 44% of the disease alleles, a heterogeneous spectrum of mutations was identified. Sixteen new, putative disease-causing mutations were detected, of which 12 were private and four were shared by unrelated patients. The disease-causing mutations were scattered throughout the gene and included six nonsense and seven missense mutations, two deletions and one small insertion. In addition, six non-pathogenic polymorphisms were identified. All missense mutations resulted in major amino acid side-chain alterations. Four missense mutations affected the N-terminal part of USH2A, whereas three missense mutations affected the laminin-type epidermal growth factor-like (LE) domain. The structural consequences of the mutations affecting the LE domain are discussed in relation to the three-dimensional structure of a LE-module of the mouse laminin gamma1 chain.     

Unraveling the molecular pathogenesis of free sialic acid storage disorders: altered targeting of mutant sialin

Salla disease (SD) and infantile sialic acid storage disease (ISSD) are recessively inherited, neuro-degenerative disorders caused by mutations in the SLC17A5 gene. The gene product, sialin, is a lysosomal membrane protein which transports free sialic acid across the membrane. Although the function of sialin is basically known, the details of biosynthesis and intracellular trafficking as well as functional consequences of disease mutations in the SLC17A5 gene are not characterized. Here we studied for the first time the expression, localization, and targeting of the wild-type sialin as well as two mutant polypeptides; one mimicking the Finnish founder mutation, R39C (Salla(FIN)), and the other a deletion (del268-272) found in ISSD patients using in vitro expression of the corresponding cDNA constructs. The wild-type sialin was targeted to lysosomes whereas a significant fraction of the Salla(FIN) polypeptides and the majority of the ISSD polypeptides remained in the Golgi compartment. Further, using a temperature block of intracellular transport, we observed that the rate of the trafficking of the mutant polypeptides to lysosomes is significantly slower than that of their wild-type counterpart. These findings are in line with the phenotypic differences between SD and ISSD, the former presenting mental retardation with long life span in contrast to the latter being an early fatal disorder.     

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates.     

Defective secretion of recombinant fragments of fibrillin-1: implications of protein misfolding for the pathogenesis of Marfan syndrome and related disorders

Fibrillin-1 is a large modular glycoprotein that assembles to form 10-12 nm microfibrils in the extracellular matrix. Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome and related connective tissue disorders (fibrillinopathies) that show autosomal dominant inheritance. The pathogenic mechanism is thought to be a dominant negative effect of a mutant protein on microfibril assembly, although direct evidence is lacking. A significant group of disease-causing FBN1 mutations are cysteine substitutions within EGF domains that are predicted to cause misfolding by removal of disulphide bonds that stabilize the native domain fold. We have studied three missense mutations (C1117Y, C1129Y and G1127S) to investigate the effect of misfolding on the trafficking of fibrillin-1 from fibroblast cells. We demonstrate that both C1117Y and C1129Y, expressed as recombinant fragments of fibrillin-1, are retained and accumulate within the cell. Both undergo core glycosylation but lack the complex glycosylation observed in the secreted wild-type fragment, suggesting retention in the endoplasmic reticulum (ER). In addition, co-immunoprecipitation experiments show association with the ER chaperone calreticulin, but not calnexin, 78 kDa glucose-regulated protein (Grp78/BiP) or protein disulfide isomerase. In contrast, G1127S, which causes a moderate change in the EGF domain fold, shows a pattern of glycosylation and trafficking profile indistinguishable from the wild-type fragment. Since expression of the recombinant fragments does not disrupt the secretion of endogenous fibrillin-1 by the cell, we propose that G1127S causes disease via an extracellular dominant negative effect. In contrast, the observed ER retention of C1117Y and C1129Y suggests that disease associated with these missense mutations is caused either by an intracellular dominant negative effect or haploinsufficiency.     

Inhibition of protein kinase A activity interferes with long-term,but not short-term,memory of conditioned taste aversions

The present experiments examined whether inhibition of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity interferes with conditioned taste aversion (CTA) memories. Rats were centrally infused with the selective PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) before conditioning. Direct infusions of Rp-cAMPS into the amygdala showed no interference with short-term memory but did show significant attenuation of long-term memory and more rapid extinction. Results suggest that PKA activity is involved in the consolidation of long-term memory of CTAs, and that the amygdala may be 1 site that is important for this activity.     

LRRK2 as a negative regulator of NFAT: implications for the pathogenesis of inflammatory bowel disease

Inflammatory bowel disease encompasses two chronic, complex relapsing diseases believed to be caused by an excessive and poorly controlled immune response to the intestinal microbiota. The advent of genome-wide association scans has allowed for rapid identification of multiple susceptibility genes for Crohn's disease and ulcerative colitis. A locus containing LRRK2 and MUC19 is a confirmed Crohn's disease susceptibility locus. This genetic association precipitates an important functional study in which Liu et al. demonstrate that: LRRK2 acts as a negative regulator of NFAT in an NRON-dependent manner; LRRK2-deficient mice exhibit increased susceptibility to the development of dextran sodium sulfate colitis, which is enhanced by NFAT translocation to the nucleus; and patients exhibiting the risk LRRK2 allele have lower LRRK2 protein levels and exhibit less inhibition of NFAT. These data support the concept that defects within innate immunity trigger a robust adaptive immune response, which results in the production of proinflammatory cytokines and the development of chronic inflammation of the gastrointestinal mucosa.