Activation of endoplasmic reticulum-specific stress responses associated with the conformational disease Z alpha 1-antitrypsin deficiency

Conformational diseases are a class of disorders associated with aberrant protein accumulation in tissues and cellular compartments. Z alpha1-antitrypsin (A1AT) deficiency is a genetic disease associated with accumulation of misfolded A1AT in the endoplasmic reticulum (ER) of hepatocytes. We sought to identify intracellular events involved in the molecular pathogenesis of Z A1AT-induced liver disease using an in vitro model system of Z A1AT ER accumulation. We investigated ER stress signals induced by Z A1AT and demonstrated that both the ER overload response and the unfolded protein response were activated by mutant Z A1AT, but not wild-type M A1AT. Interestingly, activation of the unfolded protein response pathway required an additional insult, whereas NF-kappa B activation, a hallmark of the ER overload response, was constitutive. These findings have important implications for the design of future therapeutics for Z A1AT liver disease and may also impact on drug design for other conformational diseases.     

Polymorphisms of hemochromatosis, and alpha-1 antitrypsin genes in Egyptian HCV patients with and without hepatocellular carcinoma

Hereditary hemochromatosis and alpha-1antitrypsin deficiency are genetic diseases characterized by endoplasmic reticulum (ER) stress with subsequent development of liver disease. Our aim was to estimate the frequency of hemochromatosis gene (HFE) mutant alleles (C282Y and H63D) and alpha-1 antitrypsin S/Z variants among Egyptian HCV cirrhotic patients and in hepatocellular carcinoma patients and to evaluate their effects on disease progression. HFE and alpha-1 antitrypsin polymorphisms were characterized in 200 Egyptian patients with HCV infection (100 patients complicated with cirrhosis, 100 patients with HCC) and 100 healthy subjects who had no history of any malignancy. The frequencies of HD genotype of H63D mutation were significantly increased in HCC patients compared to control group and to cirrhosis group. Also, the frequencies of DD genotype were significantly increased In HCC group compared to control group and to cirrhosis group. Our results suggested that Carriers of the D allele of H63D mutation were significantly more likely to develop HCC.     

A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA.     

The effect of neuraminidase on genetic variants of alpha anitrypsin.

Sera of Pi types M, F, S, Z, IM, FM, MS, and MZ were incubated with neuraminidase and the reaction products followed by electrophoresis. The alpha1 antitrypsin components showed a series of changes in mobility as sialic residues were removed. Removal of sialic acid was confirmed by chemical assay. Results of studies with two different electrophoretic systems suggested that the Z type alpha1 antitrypsin has less sialic acid than the M, F, and S types. There was no evidence that other genetic variants have a reduced sialic acid content. The two major bands of alpha1 antitrypsin seen in certain electrophoretic systems may reflect a difference of one sialic acid residue. It is proposed that the Z protein lacks a carbohydrate chain with two terminal sialic acid residues. This carbohydrate deficiency results in lack of secretion of type Z alpha1 antitrypsin from the endoplasmic reticulum, perhaps because of binding to sites specific for the incomplete glycoprotein or because of aggregation of the Z asialo protein. A carbohydrate chain could be prevented from attaching to the Z type either because of a conformational change or because of the replacement of a carbohydrate-binding asparagine residue in the Z protein.     

Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.     

Glucosidase and mannosidase inhibitors mediate increased secretion of mutant alpha1 antitrypsin Z

It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein alpha1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of alpha1 antitrypsin (alpha1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of alpha1 antitrypsin Z (alpha1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of alpha1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with alpha1-AT deficiency.     

Regulator of G Signaling 16 is a marker for the distinct endoplasmic reticulum stress state associated with aggregated mutant alpha1-antitrypsin Z in the classical form of alpha1-antitrypsin deficiency

In the classical form of alpha(1)-antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant alpha(1)-antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble alpha(1)-antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic alpha(1)-antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with alpha(1)-antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble alpha(1)-antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in alpha(1)-antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.     

Selenoprotein S/SEPS1 Modifies Endoplasmic Reticulum Stress in Z Variant ��1-Antitrypsin Deficiency

Z α₁-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFκB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFκB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFκB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.SEPS1 (selenoprotein S, VIMP, Tanis, or SelS) is a selenoprotein found in the endoplasmic reticulum (ER)³ membrane. SEPS1 participates in the processing and removal of misfolded proteins from the ER to the cytosol, where they are polyubiquitinated and degraded through the proteasome (1). SEPS1 can be induced by ER stress (2) and has been shown in macrophages to be protective from pharmacological ER stress agent-induced apoptosis (3).The endoplasmic reticulum is one of the largest cell organelles. It serves many essential functions, including production of all components of cellular membranes, proteins, lipids, and sterols (4). Only correctly folded proteins are transported out of the ER, whereas incompletely folded proteins are retained in the organelle to complete the folding process or be targeted for destruction. ER stress is defined as an imbalance between the cellular demand for ER function and capacity of the organelle. It is characterized by a number of intracellular responses. These responses include the ER overload response (EOR), the unfolded protein response (UPR), and apoptosis.α₁-Antitrypsin (AAT) deficiency is a disease characterized by early onset emphysema and liver disease (5). The mutant Z form of this autosomal co-dominant disease occurs in >95% of all individuals with AAT deficiency (6). Liver disease occurs in ∼10% of all homozygous neonates who develop hepatitis and cholestasis. A proportion of these children progress to liver failure, requiring liver transplantation (7, 8). Cirrhosis can also occur in adults without a preceding history of childhood liver disease. The mutant Z AAT polymerizes and accumulates in the ER, leaving only 15% of ZAAT secreted (9, 10). This accumulation of abnormal protein in the ER gives rise to ER stress, which is believed to contribute to the liver disease that results from AAT deficiency. The cells respond to this perturbation by inducing the expression of novel genes whose products aim to restore normal ER function (11). SEPS1 is an example of a molecular chaperone that serves to augment the capacity of the ER for protein folding and degradation.In this paper, we investigate the role of SEPS1 in regulating the cellular response to ER stress in HepG2 cells transfected with the ZAAT transgene. We investigate the effect of SEPS1 on the UPR component of this response by measuring grp78 promoter activity, a UPR-up-regulated gene that functions as a molecular chaperone, and by detecting activated ATF6, which occurs downstream to the activation of grp78. The EOR component of ER stress is investigated by measuring NFκB activation.We study the effect of selenium supplementation on the action of SEPS1 to see if the function of this selenoprotein can be enhanced and, if so, through which pathways, looking specifically at grp78 promoter activity, ATF6 activation, and NFκB activation and also at glutathione peroxidase (GPx) activity and at the anti-inflammatory 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) pathway. GPx is a selenoprotein whose activity can be readily assayed. This was used as a measure of selenoprotein activity.The role of SEPS1 in conformational diseases has not been evaluated. These diseases are caused by inherited or acquired modifications in protein structure, where specific proteins undergo a conformational rearrangement, causing deposition within cellular compartments, such as the ER. This can lead to devastating results. AAT deficiency is one such disease, but the group includes Alzheimer, Parkinson, and Huntington diseases and cystic fibrosis.     

Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Tris reversibly inhibits secretion of albumin and alpha-1-antitrypsin at different sites

We have investigated the effect of the weak base Tris on the processing and secretion of albumin and alpha-1 antitrypsin by hepatocytes in culture. We show that the secretion of both proteins is 90% inhibited by 30 mM Tris. The post-synthetic processing of both proteins is inhibited to the same extent. These effects are completely reversible. Cell fractionation indicates that albumin accumulates in the Golgi, whereas alpha-1 antitrypsin fails to leave the endoplasmic reticulum.     

Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency

Homozygous, PIZZ alpha(1)-antitrypsin (alpha(1)-AT) deficiency is associated with chronic liver disease and hepatocellular carcinoma resulting from the toxic effects of mutant alpha(1)-anti-trypsin Z (alpha(1)-ATZ) protein retained in the endoplasmic reticulum (ER) of hepatocytes. However, the exact mechanism(s) by which retention of this aggregated mutant protein leads to cellular injury are still unknown. Previous studies have shown that retention of mutant alpha(1)-ATZ in the ER induces an intense autophagic response in hepatocytes. In this study, we present evidence that the autophagic response induced by ER retention of alpha(1)-ATZ also involves the mitochondria, with specific patterns of both mitochondrial autophagy and mitochondrial injury seen in cell culture models of alpha(1)-AT deficiency, in PiZ transgenic mouse liver, and in liver from alpha(1)-AT-deficient patients. Evidence for a unique pattern of caspase activation was also detected. Administration of cyclosporin A, an inhibitor of mitochondrial permeability transition, to PiZ mice was associated with a reduction in mitochondrial autophagy and injury and reduced mortality during experimental stress. These results provide evidence for the novel concept that mitochondrial damage and caspase activation play a role in the mechanism of liver cell injury in alpha(1)-AT deficiency and suggest the possibility of mechanism-based therapeutic interventions.     

ADD66, a gene involved in the endoplasmic reticulum-associated degradation of alpha-1-antitrypsin-Z in yeast, facilitates proteasome activity and assembly

Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the "Z" variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an approximately 30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Delta mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD.     

内质网应激反应分子机理研究进展

内质网应激是导致心脑组织缺血梗塞、神经退行性疾病等发生的重要环节 .目前发现同型半胱氨酸、氧化应激、钙代谢紊乱等都能引起内质网应激级联反应 ,表现为蛋白质合成暂停、内质网应激蛋白表达和细胞凋亡等 .这些表现包括在未折叠蛋白反应 (UPR)、整合应激反应 (ISR)和内质网相关性死亡 (ERAD)三个相互关联的动态过程中 ,每一过程的分子机理现已逐步被揭示 .作为细胞保护性应对机制的内质网应激体系一旦遭到破坏 ,细胞将不能合成应有的蛋白质 ,亦不能发挥正常的生理功能 ,甚至会出现细胞凋亡 .掌握内质网应激过程对进一步理解多种疾病的发生机理有十分重要的理论意义     

ER stress and diseases

Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells activate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and translational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorder, which are collectively known as 'conformational diseases'. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases.     

A novel model and molecular therapy for Z alpha-1 antitrypsin deficiency

Animal models that closely resemble human disease can present a challenge. Particularly so in alpha-1 antitrypsin deficiency (α(1)ATD), as the mouse alpha-1 antitrypsin (α(1)AT) cluster encodes five highly related genes compared with the one in humans. The mouse PI2 homologue is closest to the α(1)AT human gene. We have changed the equivalent mouse site that results in the Z variant in man (Glu342Lys) and made both the "M" and "Z" mouse PI2 α(1)AT proteins. We have tested the ability of a small-molecular-weight compound CG to alleviate polymerisation of these mouse α(1)AT proteins as it has been shown to reduce aggregates of Z α(1)AT in man. We found that (1) CG specifically reduces the formation of polymers of recombinant mouse "Z" protein but not "M" protein; (2) whereas there is significantly more α(1)AT secreted from Chinese Hamster Ovary cells transfected with the mouse "M" α(1)AT gene than with the "Z" (20.8?±?3.9 and 6.7?±?3.6, respectively; P?相似文献   

A naturally occurring nonpolymerogenic mutant of alpha 1-antitrypsin characterized by prolonged retention in the endoplasmic reticulum.

The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

The Role of Autophagy in Alpha-1-Antitrypsin Deficiency: A Specific Cellular Response in Genetic Diseases Associated with Aggregation-Prone Proteins

In the classical form of alpha-1-antitrypsin (AT) deficiency a point mutation renders aggregation-prone properties on a hepatic secretory protein. The mutant ATZ protein in retained in the endoplasmic reticulum (ER) of liver cells rather than secreted into the blood and body fluids where it ordinarily functions as an inhibitor of neutrophil proteases. A loss-of-function mechanism allows the neutrophil proteases to slowly destroy the connective tissue matrix of the lung, resulting in premature development of pulmonary emphysema as early as the third decade of life. A gain-of-toxic function mechanism is responsible for liver inflammation and carcinogenesis. Indeed this deficiency is the most common genetic cause of liver disease in children in the US. It also causes chronic liver inflammation and carcinoma that manifests itself later in life. However, the majority of affected homozygotes apparently escape liver disease. This last observation has led to the concept that genetic and/or environmental modifiers affect the disposal of mutant ATZ within the ER or affect the protective cellular responses activated by accumulation of ATZ in the ER and, in turn, these modifiers determine which homozygotes develop liver inflammation and carcinoma. In this article I review a series of studies published over the last 6 years showing that autophagy is specifically activated by ER accumulation of ATZ and that autophagy plays a critical role in the disposal of this mutant protein. Further, I review data suggesting that the autophagy is specifically designed for the cellular response to aggregated ATZ and aggregated proteins in general.