Deficiency of Aph1B/C-gamma-secretase disturbs Nrg1 cleavage and sensorimotor gating that can be reversed with antipsychotic treatment

Regulated intramembrane proteolysis by gamma-secretase cleaves proteins in their transmembrane domain and is involved in important signaling pathways. At least four different gamma-secretase complexes have been identified, but little is known about their biological role and specificity. Previous work has demonstrated the involvement of the Aph1A-gamma-secretase complex in Notch signaling, but no specific function could be assigned to Aph1B/C-gamma-secretase. We demonstrate here that the Aph1B/C-gamma-secretase complex is expressed in brain areas relevant to schizophrenia pathogenesis and that Aph1B/C deficiency causes pharmacological and behavioral abnormalities that can be reversed by antipsychotic drugs. At the molecular level we find accumulation of Nrg1 fragments in the brain of Aph1BC(-/-) mice. Our observations gain clinical relevance by the demonstration that a Val-to-Leu mutation in the Nrg1 transmembrane domain, associated with increased risk for schizophrenia, affects gamma-secretase cleavage of Nrg1. This finding suggests that dysregulation of intramembrane proteolysis of Nrg1 could increase risk for schizophrenia and related disorders.     

Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion

Gliding motility and host-cell invasion by apicomplexan parasites depend on cell-surface adhesins that are translocated via an actin–myosin motor beneath the membrane. The current model posits that fructose-1,6-bisphosphate aldolase (ALD) provides a critical link between the cytoplasmic tails of transmembrane adhesins and the actin–myosin motor. Here we tested this model using the Toxoplasma gondii apical membrane protein 1 (TgAMA1), which binds to aldolase in vitro. TgAMA1 cytoplasmic tail mutations that disrupt ALD binding in vitro showed no correlation with host-cell invasion, indicating this interaction is not essential. Furthermore, ALD-depleted parasites were impaired when grown in glucose, yet they showed normal gliding and invasion in glucose-free medium. Depletion of ALD in the presence of glucose led to accumulation of fructose-1,6-bisphosphate, which has been associated with toxicity in other systems. Finally, TgALD knockout parasites and an ALD mutant that specifically disrupts adhesin binding in vitro also supported normal invasion when cultured in glucose-free medium. Taken together, these results suggest that ALD is primarily important for energy metabolism rather than interacting with microneme adhesins, challenging the current model for apicomplexan motility and invasion.The phylum Apicomplexa is a group of mostly intracellular parasites that contains a number of human pathogens, including Toxoplasma gondii and Plasmodium spp., the causative agent of malaria. As intracellular pathogens, efficient host-cell invasion is critical for survival, dissemination, and transmission of these parasites. Although they infect different types of host cells, apicomplexan parasites share a conserved mode of host-cell invasion that relies on regulated secretion of adhesive proteins and active motility that is powered by an actin–myosin motor complex (1, 2). According to the current model, motility and host-cell invasion depend on transmembrane adhesins that are secreted apically from the micronemes and translocated along the cell surface in a conveyor belt fashion, using the force generated by the motor complex beneath the parasite membrane (1, 2).Micronemal adhesins contain a variety of extracellular adhesive domains, a transmembrane domain, and a short cytoplasmic tail that is rich in acidic residues and contains a tryptophan residue (Trp) at or near the extreme carboxyl terminus (3). These conserved features of the cytoplasmic tails are critical to their function, as shown by mutational studies and functional replacement of the thrombospondin-related adhesive protein (TRAP) tail (TRAPt) in P. berghei with the T. gondii Microneme Protein 2 (MIC2) tail (TgMIC2t) (4). The cytoplasmic tails of several micronemal adhesins are thought to be anchored to the actin–myosin motor through a bridging molecule, the glycolytic enzyme fructose-1,6-bisphosphate aldolase (ALD) (5, 6). Mutational analysis has shown that in vitro binding of TgMIC2t and PbTRAPt to ALD is mediated by the acidic residues and Trp residue in the adhesin tails (5–7). ALD has also been shown to interact with MIC2 and TRAP in coimmunoprecipitation experiments using parasite lysates (5, 6). Further support that this interaction plays a role in vivo comes from a conditional knockout (cKO) of TgALD, which shows impaired invasion and growth, consistent with a role in metabolism and/or bridging of adhesins (8). Evidence that TgALD plays a specific role in bridging to adhesins was provided by the TgALD mutant K41E-R42G, which dramatically reduces TgMIC2t binding in vitro whereas having a minimal effect on enzyme activity (8). When expressed in the conditional knockout strain of TgALD (ALD cKO), the K41E-R42G mutant has normal ATP levels, yet it shows decreased host-cell invasion (8).The micronemal adhesin AMA1 is also important for host-cell invasion by T. gondii and Plasmodium spp. (9–11). AMA1 has similar topology with TRAP/MIC2 with its bulky ectodomain binding to rhoptry neck proteins (RONs) that are secreted from the rhoptries and inserted into host plasma membrane to mediate formation of a moving junction between the host and parasite membranes (12–15). The cytoplasmic tail of AMA1 (AMA1t) also binds rabbit ALD in vitro, and a TgAMA1 mutant with a pair of aromatic residues changed to alanines (i.e., F547A, W548A) disrupts aldolase binding in vitro and blocks host-cell invasion (16). Similarly located FW residues in P. falciparum AMA1t were also shown to mediate binding to rabbit aldolase in vitro, implying this interaction is conserved (17). These studies suggest that AMA1 binding to ALD may play a key role during parasite invasion, similar to that proposed for MIC2 (8) and TRAP (6).In the present study, we undertook a broader analysis of TgAMA1t mutants that disrupt binding to TgALD in vitro to determine the role of this interaction during host-cell invasion. Unexpectedly, our results indicate that the TgALD–TgAMA1 interaction is not required for parasite motility or invasion. Moreover, we found that the previously described role for TgALD during invasion is alleviated by the absence of glucose. Taken together, these results suggest that ALD is primarily important for energy metabolism but does not play an essential role in coupling adhesins to the motor complex during invasion.     

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma

Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.     

Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate

Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure-function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.     

Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by site-2 protease

The NH(2)-terminal domains of membrane-bound sterol regulatory element-binding proteins (SREBPs) are released into the cytosol by regulated intramembrane proteolysis, after which they enter the nucleus to activate genes encoding lipid biosynthetic enzymes. Intramembrane proteolysis is catalyzed by Site-2 protease (S2P), a hydrophobic zinc metalloprotease that cleaves SREBPs at a membrane-embedded leucine-cysteine bond. In the current study, we use domain-swapping methods to localize the residues within the SREBP-2 membrane-spanning segment that are required for cleavage by S2P. The studies reveal a requirement for an asparagine-proline sequence in the middle third of the transmembrane segment. We propose a model in which the asparagine-proline sequence serves as an NH(2)-terminal cap for a portion of the transmembrane alpha-helix of SREBP, allowing the remainder of the alpha-helix to unwind partially to expose the peptide bond for cleavage by S2P.     

Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx

Plasmodium falciparum apical membrane antigen 1 (AMA1) is located in the merozoite micronemes, an organelle that contains receptors for invasion, suggesting that AMA1 may play a role in this process. However, direct evidence that P. falciparum AMA1 binds to human erythrocytes is lacking. In this study, we determined that domain III of AMA1 binds to the erythrocyte membrane protein, Kx, and that the rate of invasion of Kx(null) erythrocytes is reduced, indicating a significant but not unique role of AMA1 and Kx in parasite invasion of erythrocytes. Domains I/II/III, domains I/II and domain III of AMA1 were expressed on the surface of CHO-K1 cells, and their ability to bind erythrocytes was determined. We observed that each of these domains failed to bind untreated human erythrocytes. In contrast, domain III, but not the other domains of AMA1, bound to trypsin-treated human erythrocytes. We tested the binding of AMA1 to trypsin-treated genetically mutant human erythrocytes, missing various erythrocyte membrane proteins. AMA1 failed to bind trypsin-treated Kx(null) (McLeod) erythrocytes, which lack the Kx protein. Furthermore, treatment of human erythrocytes with trypsin, followed by alpha-chymotrypsin, cleaved Kx and destroyed the binding of AMA1 to human erythrocytes. Lastly, the rate of invasion of Kx null erythrocytes by P. falciparum was significantly lower than Kx-expressing erythrocytes. Taken together, our data suggest that AMA1 plays an important, but not exclusive, role in invasion of human erythrocytes through a process that involves exposure or modification of the erythrocyte surface protein, Kx, by a trypsin-like enzyme.     

Immunization with a functional protein complex required for erythrocyte invasion protects against lethal malaria

An essential step in the invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites is the binding of rhoptry neck protein 2 (RON2) to the hydrophobic groove of apical membrane antigen 1 (AMA1), triggering junction formation between the apical end of the merozoite and the RBC surface to initiate invasion. Vaccination with AMA1 provided protection against homologous parasites in one of two phase 2 clinical trials; however, despite its ability to induce high-titer invasion-blocking antibodies in a controlled human challenge trial, the vaccine conferred little protection even against the homologous parasite. Here we provide evidence that immunization with an AMA1-RON2 peptide complex, but not with AMA1 alone, provided complete protection against a lethal Plasmodium yoelii challenge in mice. Significantly, IgG from mice immunized with the complex transferred protection. Furthermore, IgG from PfAMA1-RON2–immunized animals showed enhanced invasion inhibition compared with IgG elicited by AMA1 alone. Interestingly, this qualitative increase in inhibitory activity appears to be related, at least in part, to a switch in the proportion of IgG specific for certain loop regions in AMA1 surrounding the binding site of RON2. Antibodies induced by the complex were not sufficient to block the FVO strain heterologous parasite, however, reinforcing the need to include multiallele AMA1 to cover polymorphisms. Our results suggest that AMA1 subunit vaccines may be highly effective when presented to the immune system as an invasion complex with RON2.Malaria caused by Plasmodium falciparum (Pf) remains one of the leading causes of mortality in pregnant women and children in sub-Saharan Africa (1). The lack of a vaccine and resistance to front-line antimalarials pose a global public health threat. RTS,S, a leading vaccine candidate that targets the initial infection of the liver, has demonstrated only partial efficacy (2). Clinical manifestations of malaria are caused by the blood-stage parasites that reside within red blood cells (RBCs); thus, vaccines targeting the erythrocytic forms of the parasite are desirable for efficient disease control.Apical membrane antigen 1 (AMA1) was once considered a leading blood-stage vaccine candidate, because antibodies against recombinant AMA1 are highly efficient in blocking entry of parasites into RBCs both in vitro and in immunized nonhuman primates in vivo (3, 4). Disappointingly, however, despite these early successes, moderate to no efficacy was observed in human trials (5–7). Previous in vitro assays have shown that AMA1 polymorphisms among different parasite strains rendered the antibodies allele-specific (8, 9); however, the failure in a controlled human clinical trial cannot be attributed to polymorphisms, given that the vaccine was not efficient even against homologous parasites (5). Although the vaccinations failed to induce immunity against the homologous parasite in vivo, AMA1-specific antibodies purified from these individuals blocked parasite invasion in vitro (5).Two phase 2 trials have been reported to date (6, 7). Ouattara et al. (6) reported no significant efficacy of a bivalent AMA1 vaccine adjuvanted with aluminum hydroxide even against homologous parasites, whereas Thera et al (7), in a study of a monovalent AMA1 vaccine with a different adjuvant system, found 64% efficacy against vaccine-type allele as defined by amino acid homology in the domain Id loop (cluster 1L). The number of vaccine-type parasites in that study population was very low (∼5%), however, and whether this level of efficacy can be achieved in a larger sample size remains to be determined. That study also confirmed the importance of polymorphisms in AMA1 contributing to lack of efficacy against heterologous parasites (7, 10). Recent efforts to cover the polymorphism in AMA1 demonstrated that combining four or five different AMA1 alleles could overcome the strain-specific barrier in vitro (9, 11, 12). Nonetheless, the discordance between failure to protect humans in vivo and ability to block vaccine-type parasite invasion in vitro (5) underscores the need to improve AMA1 vaccine efficacy against homologous parasites.It was recently reported that AMA1 interacts with a conserved 49-aa region of RON2, a parasite rhoptry-resident protein, during merozoite invasion (13–15). Small molecules or peptides that block this interaction inhibit merozoite invasion (16, 17), highlighting the important role of this protein–protein interaction. Analysis of the crystal structure of the complex revealed that the RON2 peptide (RON2L) binds to a conserved hydrophobic groove in AMA1, resulting in extensive conformational changes in certain loop regions surrounding the groove (18, 19). Antibodies that bind in or near the hydrophobic groove block parasite invasion by inhibiting the binding of RON2 (11, 15).In this study, we evaluated a novel approach to enhancing the efficacy of the AMA1 vaccine using a highly virulent Plasmodium yoelli YM (PyYM) mouse model. Strikingly, all animals immunized with the AMA1-RON2L complex, but not those immunized with the individual antigens, were found to be protected against the virulent homologous PyYM challenge. Antibodies largely mediate this protection, as demonstrated by the fact that passive transfer of IgG, but not of T cells, from AMA1-RON2L–vaccinated animals controlled parasitemia. Furthermore, we found that the human parasite Pf3D7-AMA1-RON2L complex induced qualitatively higher growth inhibitory antibodies than AMA1 alone in in vitro assays. Surprisingly, our results indicate that the increase in inhibitory antibodies generated by the complex may be related in part to a switch in the proportion of antibodies against the loops surrounding the hydrophobic groove with which RON2 interacts. Our data suggest that a vaccine comprising a multiallele AMA1 in complex with RON2L may be more efficacious than AMA1 alone in targeting both homologous and heterologous parasites.     

The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase

Notch signaling is involved in numerous cell fate decisions in invertebrates and vertebrates. The Notch receptor is a type I transmembrane (TM) protein that undergoes two proteolytic steps after ligand binding, first by an ADAM (a distintegrin and metalloprotease) in the extracellular region, followed by gamma-secretase-mediated cleavage inside the TM domain. We demonstrate here that the murine ligand Delta1 (Dll1) undergoes the same sequence of cleavages, in an apparently signal-independent manner. Identification of the ADAM-mediated shedding site localized 10 aa N-terminal to the TM domain has enabled us to generate a noncleavable mutant. Kuzbanian/ADAM10 is involved in this processing event, but other proteases can probably substitute for it. We then show that Dll1 is part of a high-molecular-weight complex containing presenilin1 and undergoes further cleavage by a gamma-secretase-like activity, therefore releasing the intracellular domain that localizes in part to the nucleus. Using the shedding-resistant mutant, we demonstrate that this gamma-secretase cleavage depends on prior ectodomain shedding. Therefore Dll1 is a substrate for regulated intramembrane proteolysis, and its intracellular region possibly fulfills a specific function in the nucleus.     

The γ-secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus

Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease. The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma). This processing resembles regulated intramembrane proteolysis of transmembrane proteins such as Notch, where the released cytoplasmic fragments enter the nucleus and modulate gene expression. Here, we examined whether the analogous Cgamma fragments of APP also exert effects in the nucleus. We find that ectopically expressed Cgamma is present both in the cytoplasm and in the nucleus. Interestingly, expression of Cgamma59 causes disappearance of PAT1, a protein that interacts with the APP cytoplasmic domain, from the nucleus and induces its proteosomal degradation. Treatment of cells with lactacystin prevents PAT1 degradation and retains its nuclear localization. By contrast, Cgamma57, a minor product of gamma-cleavage, is only marginally effective in PAT1 degradation. Furthermore, Cgamma59 but not Cgamma57 potently represses retinoic acid-responsive gene expression. Thus, our studies provide the evidence that, as predicted by the regulated intramembrane proteolysis mechanism, Cgamma seems to function in the nucleus.     

Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.     

Presenilin-1 mutations of leucine 166 equally affect the generation of the Notch and APP intracellular domains independent of their effect on Abeta 42 production

The Alzheimer's disease (AD)-associated presenilin (PS) proteins are required for the gamma-secretase cleavages of the beta-amyloid precursor protein and the site 3 (S3) protease cleavage of Notch. These intramembrane cleavages release amyloid-beta peptide (Abeta), including the pathogenic 42-aa variant (Abeta(42)), as well as the beta-amyloid precursor protein and the Notch intracellular domains (AICD, NICD). Whereas Abeta is generated by endoproteolysis in the middle of the transmembrane domain, AICD and NICD are generated by cleavages at analogous positions close to the cytoplasmic border of the transmembrane domain. Numerous mutations causing familial AD (FAD) that all cause increased production of Abeta(42) have been found in the PS1 gene. Here we have investigated the previously uncharacterized, very aggressive FAD mutation L166P that causes onset of AD in adolescence. Strikingly, the PS1 L166P mutation not only induces an exceptionally high increase of Abeta(42) production but also impairs NICD production and Notch signaling, as well as AICD generation. Thus, FAD-associated PS mutants cannot only affect the generation of NICD, but also that of AICD. Moreover, further analysis with artificial L166 mutants revealed that the gamma-secretase cleavage at position 40/42 and the S3-like gamma-secretase cleavage at position 49 of the Abeta domain are both differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect the generation of NICD and AICD in a similar manner, supporting the concept that S3 protease and S3-like gamma-secretase cleavages are mediated by identical proteolytic activities.     

Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket

Apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate that possesses polymorphisms that may pose a problem for a vaccine based on this antigen. Knowledge of the distribution of the polymorphic sites on the surface of AMA1 is necessary to obtain a detailed understanding of their significance for vaccine development. For this reason we have sought to determine the three-dimensional structure of AMA1 using x-ray crystallography. The central two-thirds of AMA1 is relatively conserved among Plasmodium species as well as more distantly related apicomplexan parasites, and contains two clusters of disulfide-bonded cysteines termed domains I and II. The crystal structure of this fragment of AMA1 reported here reveals that domains I+II consists of two intimately associated PAN domains. PAN domain I contains many long loops that extend from the domain core and form a scaffold for numerous polymorphic residues. This extreme adaptation of a PAN domain reveals how malaria parasites have introduced significant flexibility and variation into AMA1 to evade protective human antibody responses. The polymorphisms on the AMA1 surface are exclusively located on one side of the molecule, presumably because this region of AMA1 is most accessible to antibodies reacting with the parasite surface. Moreover, the most highly polymorphic residues surround a conserved hydrophobic trough that is ringed by domain I and domain II loops. Precedents set by viral receptor proteins would suggest that this is likely to be the AMA1 receptor binding pocket.     

DPYD、ABCB1、GSTP1、ERCC1基因多态性与晚期结肠癌临床特征、不良反应、预后的关系

目的检测晚期肠癌患者药物基因多态性,分析其与临床特征、以奥沙利铂或氟尿嘧啶为主化疗方案不良反应和预后的关系。 方法收集2016年3月至2018年5月在中国解放军总医院肿瘤内科住院治疗的108例晚期结肠癌患者的外周血,以Life平台检测对DPYD、ABCB1、GSTP1、ERCC1基因进行单核苷酸多态性（SNP）分型,比较不同基因型与患者KRAS状态、肿瘤部位（左右）、不良反应和中位无进展生存时间（PFS）的差异。 结果纳入晚期肠癌患者108例,DPYD 4个位点（rs3918290、rs55886062、rs67376798、rs2297595）均为野生型,ERCC1（rs11615）GG纯合型基因52例（48.1%）,AG杂合型基因50例（46.3%）,AA野生型基因6例（5.6%）。GSTP1（rs1695）AG杂合型突变36例（33.3%）,AA野生型66例（66.1%）,GG纯和突变型6例（5.6%）。ABCB1（rs1045642）AG杂合型基因58例（53.7%）,GG纯合型基因42例（38.9%）,AA野生型8例（7.4%）。肿瘤位于左右半结肠与ERCC1基因分布频率有关（χ²=4.802,P=0.028）,与GSTP1,ABCB1基因分布频率无关。KRAS突变患者ABCB1杂合突变率42.9%,未见突变患者为72.7%,两者差异具有统计学意义（χ²=3.939,P=0.047）。GSTP1 AG型和GG型较AA型易产生2~3级全身不良反应高（77.8% vs. 45.5%,χ²=5.193;P=0.023）。ABCB1 GG型和AA型患者发生3~4级不良反应率为32.9%,对比AG型患者发生不良反应率为62.1%,差异具有统计学意义（χ²=4.862,P=0.027）。中位疾病进展时间PFS与ABCB1和GSTP1基因多态性无关,与不同ERCC1基因型有关,ERRC1杂合型突变（AG型）患者较纯和型（GG型＋AA型）具有较短PFS（5.6 m vs. 8.0 m,P=0.029）。 结论检测基因多态性具有临床价值,对晚期肠癌化疗的不良反应、预后及为患者调整化疗方案具有有效的指导作用。     

The initial substrate-binding site of gamma-secretase is located on presenilin near the active site

gamma-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid beta-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid beta-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.     

Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum.

The insertion of proteins into the endoplasmic reticulum is mediated by short hydrophobic domains called signal sequences, which are usually cleaved during insertion. We previously constructed DNAs encoding vesicular stomatitis virus glycoproteins with N-terminal extensions preceding the signal sequence and showed that these extensions allowed normal signal-sequence function and cleavage in vivo. To analyze signal sequence topology during membrane insertion, we generated a point mutation that blocks cleavage of these signal sequences. After expressing these proteins in HeLa cells, we used proteolysis of microsomal membranes to determine that the N terminus of the signal sequence and the C terminus of each protein remain on the cytoplasmic side of the endoplasmic reticulum after insertion. This result indicates that the proteins were inserted in a looped configuration. Extending this finding, we were able to reverse the orientation of such a mutant protein by deleting its normal C-terminal transmembrane and cytoplasmic domains. In addition to demonstrating that a signal sequence can function as a membrane anchor, these findings show that except for the presence of a cleavage site, the cleaved signal sequence of a type I transmembrane protein is structurally and functionally equivalent to the noncleaved signal sequences of type II transmembrane proteins.     

Interacting alleles of the coinhibitory immunoreceptor genes cytotoxic T-lymphocyte antigen 4 and programmed cell-death 1 influence risk and features of primary biliary cirrhosis

Autoimmune diseases such as primary biliary cirrhosis (PBC) result from failure in the immune mechanisms that establish and maintain self-tolerance. Evidence suggests that these processes are shared among the spectrum of autoimmune syndromes and are likely genetically determined. Cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell-death 1 (PDCD1) are two genes encoding coinhibitory immunoreceptors that harbor polymorphisms with demonstrated associations to multiple autoimmune disorders. We aimed to assess functional single nucleotide polymorphisms (SNPs) in these two genes for association with PBC. SNPs in CTLA4 and PDCD1 were genotyped in 351 PBC patients and 205 controls. Allele and genotype frequencies were evaluated for association with PBC and/or antimitochondrial antibody (AMA) positivity with logistic regression. Haplotypes were inferred with an expectation-maximization algorithm, and allelic interaction was analyzed by logistic regression modeling. Individual SNPs demonstrated no association to PBC. However, the GG genotype of CTLA4 49AG was significantly associated with AMA positivity among the PBC patients. Also, individual SNPs and a haplotype of CTLA4 as well as a rare genotype of the PDCD1 SNP PD1.3 were associated with orthotopic liver transplantation. As well, we identified the influence of an interaction between the putatively autoimmune-protective CTLA4 49AG:CT60 AA haplotype and autoimmune-risk PDCD1 PD1.3 A allele on development of PBC. CONCLUSION: Our findings illustrate the complex nature of the genetically induced risk of PBC and emphasize the importance of considering definable subphenotypes of disease, such as AMA positivity, or definitive measures of disease severity/progression, like orthotopic liver transplantation, when genetic analyses are being performed. Comprehensive screening of genes involved with immune function will lead to a greater understanding of the genetic component of autoimmunity in PBC while furthering our understanding of the pathogenic properties of this enigmatic disease.     

Virulence-transmission trade-offs and population divergence in virulence in a naturally occurring butterfly parasite

Why do parasites harm their hosts? Conventional wisdom holds that because parasites depend on their hosts for survival and transmission, they should evolve to become benign, yet many parasites cause harm. Theory predicts that parasites could evolve virulence (i.e., parasite-induced reductions in host fitness) by balancing the transmission benefits of parasite replication with the costs of host death. This idea has led researchers to predict how human interventions—such as vaccines—may alter virulence evolution, yet empirical support is critically lacking. We studied a protozoan parasite of monarch butterflies and found that higher levels of within-host replication resulted in both higher virulence and greater transmission, thus lending support to the idea that selection for parasite transmission can favor parasite genotypes that cause substantial harm. Parasite fitness was maximized at an intermediate level of parasite replication, beyond which the cost of increased host mortality outweighed the benefit of increased transmission. A separate experiment confirmed genetic relationships between parasite replication and virulence, and showed that parasite genotypes from two monarch populations caused different virulence. These results show that selection on parasite transmission can explain why parasites harm their hosts, and suggest that constraints imposed by host ecology can lead to population divergence in parasite virulence.     

贵州省地方性氟中毒地区人群GSTP1基因Ile105Val位点多态性分析

目的 探讨贵州省毕节市燃煤型氟中毒地区人群谷胱甘肽硫转移酶(GSTs)活性及GSTP1基因Ile105Val位点多态性.方法 在贵州省毕节市鸭池镇燃煤型氟中毒非改灶村抽取160名村民作为非干预组,在毕节市长春镇燃煤型氟中毒改灶村抽取153名村民作为干预组,在非病区长顺县白云山镇抽取151名村民作对照组,用比色法分别测定各组GSTs活力;PCR-限制性片段长度多态性(PCR-RFLR)法检测各组GSTP1基因Ile105Val位点多态性[野生纯合型(AA)、突变杂合型(AG)、突变纯合型(GG)].结果 GSTs活力组间比较差异有统计学习意义(F=51.71,P<0.05).其中对照组[(24.30±6.27)kU/L]高于干预组[(20.78±6.20)kU/L]、非干预组[(12.44±4.97)kU/L],组间两两比较差异有统计学意义(P均<0.05);同组性别间比较差异无统计学意义(P均>0.05).GSTP1基因Ile105Val位点多态性,干预组[AA:67.3%(103/153),AG:29.4%(45/153),GG:3.3%(5/153)]、非干预组[AA:66.9%(107/160),AG:30%(48/160),GG:3.1%(5/160)]与对照组[AA:74.8%(113/151),AG:25.2%(38/151),GG:0(0/151)]比较,差异有统计学意义(χ2=6.04、6.07,P均<0.05);干预组与非干预组比较,差异无统计学意义(χ2=0.02,P>0.05).结论 氟中毒能导致机体GSTP1基因lle105Val基因位点的多态性发生变化,人为干预对氟的摄入可改善氟对机体的影响.