The Uzbekistan 1996 Demographic Health Survey reported 60.4% of women of reproductive age (WRA) had low hemoglobin concentrations (<120 g/L), and anemia was an important public health problem. Fortification of wheat flour was identified as an appropriate intervention to address anemia due to the ubiquitous consumption of wheat flour. A National Flour Fortification Program (NFFP) was implemented in 2005.UzDonMakhsulot (UDM) flour/grey loaf program; and consumption habits of women to investigate the dietary factors associated with anemia. Estimated anemia prevalence was 34.4% , iron depletion 47.5%  and folate deficiency 28.8% ; the effect of inflammation was minimal (4% with CRP >5 mg/L). Severe anemia was more prevalent among folate deficient than iron depleted WRA. Presence of UDM first grade flour or the grey loaf was reported in 71.3% of households. Among WRA, 32.1% were aware of UDM fortification; only 3.7% mentioned the benefits of fortification and 12.5% understood causes of anemia. Consumption of heme iron-containing food (91%) and iron absorption enhancers (97%) was high, as was the consumption of iron absorption inhibitors (95%).After 3-years of the NFFP, a national survey using large country-lot quality assurance sampling was carried out to assess iron, folate, hemoglobin and inflammation status of WRA; the coverage and knowledge of the fortified first grade The NFFP coincided with a substantial decline in the prevalence of anemia. Folate deficiency was a stronger predictor of severe anemia than iron depletion. However, the prevalence of iron depletion was high, suggesting that women are not eating enough iron or iron absorption is inhibited. Fortified products were prevalent throughout Uzbekistan, though UDM flour must be adequately fortified and monitored in the future. Knowledge of fortification and anemia was low, suggesting consumer education should be prioritized. Iron-deficiency anemia (IDA) is a significant public health problem in Uzbekistan. In 1996, a Demographic Health Survey (DHS) reported 60.4% of women of reproductive age (WRA) and 60.8% of pre-school age children (pre-SAC), between 6-59 months, had low concentrations of hemoglobin (hemoglobin <120 g/L and <110 g/L respectively) Fortification of wheat flour was identified as an appropriate public health intervention to address anemia due to the ubiquitous consumption of wheat flour . In urban areas, >90% of urban households buy bread. A separate supply chain ensures that industrial urban bakers are supplied with first grade, fortified flour for the fortified grey loaves. The pattern of bread consumption is very different in rural areas of Uzbekistan, where people bake bread at home or buy it from small ‘tandoor’ bakers; it is consequently a more difficult task to provide fortified bread to the rural poor UzDonMakhsulot (UDM), and some private mills, and on the ‘grey loaf’ made from that flour in commercial bakeries From 2001 to 2004, a program supported by the Asian Development Bank funded a pilot project in which 14 flour-mills in six provinces were equipped with fortification equipment (4). In 2005, the Republic of Uzbekistan's National Flour Fortification Program (NFFP) was implemented and executed by the Uzbekistan Ministry of Health and UNICEF. The NFFP was supported by the Global Alliance for Improved Nutrition (GAIN) and the World Bank, and received technical assistance from the US Centers for Disease Control and Prevention (CDC) and the Uzbekistan Institute of Hematology and Blood Transfusion. Building upon the Asian Development Bank project, funding from GAIN allowed expansion of production of fortified flour to an additional 34 mills. Uzbekistan produces four grades of flour: premium (extraction rate 55–60%), first (extraction rate up to 72%), second (extraction rate 78–83%) and third (extraction rate 83–95%). Since the first grade flour is consumed by 61% of the population, the NFFP targeted the fortification of ‘first grade’ flour produced by a large, state-run milling agency, KAP Complex 1, which was developed by the Kazakhstan Academy of Nutrition and used in the Asian Development Bank regional flour fortification project The selected fortificant premix was the At initiation in 2005, a monitoring and evaluation sentinel system was developed to monitor the health impact of the NFFP. Using the sentinel system, 40 households were randomly sampled within selected pilot regions In this paper, the results of the national survey are presented, assessing the iron, folate and inflammation status of non-pregnant WRA following the implementation of the NFFP. The prevalence of anemia, folate deficiency, and iron depletion are estimated, and the relationship between folate deficiency, iron depletion, and severity of anemia in this population is investigated. The success of the intervention implementation is assessed using coverage of the fortified flour and the knowledge about the fortification program among WRA. Consumption habits of the women are summarized to investigate the profile of dietary factors commonly associated with anemia in WRA in Uzbekistan, to understand other factors contributing to anemia in this population.Ethical permission was obtained from the Government of Uzbekistan with a Special Decree from the President of the Republic of Uzbekistan  and an Order of the Ministry of Health of the Republic of Uzbekistan . Inclusion in the survey was dependent on the women being willing to participate in the survey and giving written informed consent for both the interview and the phlebotomy.www.lex.uz/Pages/GetAct.aspx?lact_id=41329).The research team ensured that study participants were entered into the survey voluntarily, following the ethical principles summarized in the Belmont report (9). The informed consent form included the purpose of the survey, the steps involved, the length of interview, and the individual and public benefit of taking part. This form also emphasized the voluntary and confidential nature of the survey and made a clear statement informing each woman that she should make her own decision about participation. The women were given a chance to ask questions about the study. Those who agreed to take part signed the informed consent form and the interviewer also signed the form. The age range of eligible participants, 15 to 49 years, includes adolescents. Informed consent was administrated to adolescents in person, rather than to their parents or caretakers, based on the legislation of Republic of Uzbekistan which states: “Adolescents in the age of above fourteen years old have the right to give voluntary informed consent to undergo any medical intervention or to refuse it”  Introduction to the survey; 2) Demographic and socio-economic information; 3) Availability of fortified flour/bread at the household and testing of flour; 4) Purchase and consumption of fortified flour/bread at the household; 5) Knowledge of anemia and flour fortification; 6) Iron supplementation; 7) History of anemia; 8) Diet; 9) Reproductive history; and 10) Blood sampling for analysis of hemoglobin, serum folate, C- reactive protein (CRP) and ferritin. Before the start of the survey, two four-day parallel training sessions were conducted on the sampling method, data collection, and blood collection and storage. The questionnaire and blood sampling techniques were pre-tested in selected non-survey urban and rural locations, and results of the pre-test were incorporated into the final version of the instrument Twenty field-teams were selected, comprising one interviewer and one laboratory worker. Ten supervisors were also appointed: seven provided direct supervisory activities for the interviewers (3 teams per supervisor) and the remaining three supervised the blood sampling and processing. Fieldwork took place from 17 March – 21 April, 2008.Venous blood (10 mL) was drawn from each WRA using a Vacutainer  and kept in a cool and dark place until the end of each day. A small volume of the whole blood was used to measure the hemoglobin concentration to the nearest 1 g/L using a portable b–hemoglobin photometer . Each HemoCue was checked three times a day using three control liquids  and was considered acceptable for use only if the reading was within the range specified. Altimeters were used to measure the elevation above the sea level, to adjust hemoglobin concentrations where necessary . Hemoglobin concentrations were also adjusted for smoking status. Women with hemoglobin concentrations <120 g/L were classified as anemic (13). The remaining whole blood was centrifuged for 10 minutes at 3000 rpm; the serum was removed and stored cold (+4°C) in two tubes  at local Blood Transfusion Center. At the end of each week, the serum was transported to the Hematology and Blood Transfusion Center, Tashkent where it was stored frozen at -70°C until analysis.Serum CRP concentrations were measured using the Randox full-range immunoturbidometric assay (range of assay: 0.3 –161 mg/L) on a Randox Daytona Analyser . Three levels of specific protein controls and two high sensitivity CRP controls were run at the beginning and end of the day; intra-assay precision was <3% and inter-assay precision was <6%. A CRP concentration >5 mg/L indicated the presence of inflammation Serum ferritin concentrations were measured using the Randox immunoturbidometric assay (range of assay: 5.78–434 µg/L) also on the Randox Daytona Analyser. Three levels of specific protein controls were run twice a day; the intra-assay and inter-assay precision were <5%. A ferritin concentration <15 µg/L indicated depleted iron stores Serum samples were analyzed to determine folate concentrations, using a microbiological assay in the Department of Hematology at the Tashkent Hematology and Blood Transfusion Center. The microbiological assay was based on the methodology used by the Division of Laboratory Sciences, Nutritional Biomarker Branch at the CDC, USA, but with a slight modification because of the limitation of equipment available The survey data were analyzed at the oblast and national level using Stata v12 . Large sample linearized variance estimates were used to calculate confidence intervals (CI). Survey weights were defined as the inverse probability of a sampled individual being included in the survey. To quantify socioeconomic status (SES), a principal components analysis was used, including variables reflecting the availability of durable household goods; the first principal component was subsequently divided into quintiles to construct the SES indicator CI for the national and oblast-level estimates were set at 95% and constructed using a t-distribution. In contingency table analyses assessing the relationships between categorical covariates, the design-based Pearson Chi-square test was used, correcting the Chi-square statistic and basing inference on the F-distribution to account for the survey design In the survey, 136 SAs out of the total 199 SAs were sampled. The total sample size was 2,584 women. Descriptive statistics for the study population are shown in Anemia: The estimated mean hemoglobin concentration among WRA in Uzbekistan was 121.6 g/L , with an anemia prevalence of 34.4% , indicating a moderate public health problem in the country Prevalence of anemia varied by SES (p = 0.03), with the lowest anemia prevalence in the highest SES quintile. Prevalence of anemia was 27.8%  in the highest quintile compared to 36.1%  in the remaining four SES quintiles .Inflammation: Nationally, the mean CRP concentration was low , as was the prevalence of inflammation  et alIron status: Although there was little inflammation in the study population, ferritin concentrations were adjusted for the presence of inflammation, using the calculated factors from the meta-analysis of Thurnham Prevalence of iron depletion was marginally associated with SES (p = 0.04). The highest prevalence of iron depletion was found in the third quintile of SES ; and the lowest prevalence was found in the highest SES quintile , though this estimated prevalence was similar to the national prevalence of iron depletion (47.5%).Folate status: The estimated mean folate concentration among WRA in Uzbekistan was 11.9 nmol/L ; and the prevalence of low folate was 28.8%  , with a SES was associated with folate deficiency (p = 0.001). Estimated prevalence of folate deficiency increased monotonically with SES quintile. The prevalence of women with folate deficiency in the lowest SES category was 36.2%  and decreased to 22.4%  in the highest SES category.Iron depletion and folate deficiency were both associated with anemia (p<0.0001 for both). Estimated anemia prevalence was 20.2%  among WRA without iron depletion; and 45.8%  among WRA with iron depletion. Estimated anemia prevalence was 24.7%  among WRA without folate deficiency and 51.3%  among WRA with folate deficiency. Folate deficiency and iron depletion were also both associated with severity of anemia (p<0.0001).To investigate the relative importance of folate deficiency and iron depletion in anemia severity, a four-category indicator was constructed, with categories: no iron or folate depletion; iron depletion with no folate depletion; folate deficiency with no iron depletion; and both iron and folate depletion. Anemia severity is examined within levels of this indicator in In Nationally, 96.9%  of WRA had some type of wheat flour in the household. UDM first grade flour was identified in 59.8%  of households, with substantially lower percentages in Tashkent city  and Tashkent oblast . In 88 cases (3.5%), respondents were not able to identify the type of the flour at their households, as often the flour was purchased in packages without labels.The grey loaf was less popular than UDM flour, with only 11.5%  of households having the grey loaf at the time of the survey. However, in Tashkent city and Tashkent oblast, 72.1%  and 38.5%  of households respectively had the grey loaf in the household. Altogether, 71.3%  of households consumed either the grey loaf or home-baked bread made from UDM first grade flour the day of the survey. Consumption of fortified products varied by oblast (p = 0.001), with the lowest estimated proportion reported in Jizak  and the highest in Navoi .The qualitative iron spot test carried out at the household identified that 41.6%  of all flour tested was fortified. Fortification rates varied by oblast (p<0.001) with the highest estimates of 73.5%  in Bukhara and the lowest in Syrdarya oblast  and Tashkent city .UDM first grade flour was found more frequently in households with lower SES (p<0.001). In the lowest SES group, 65.4%  had UDM first grade flour, compared to 45.0%  in the highest group. In urban areas, UDM first grade flour was available in 42.0%  of households compared to 62.6%  in rural areas (p = 0.001).A multivariate logistic regression model was used to further examine predictors of the availability of first grade flour. The following covariates were included in the regression model: age, education, SES, urban/rural location, knowledge that UDM flour is fortified, whether the woman bakes her own bread, anemia history, and iron use history. Education was not statistically significant in the regression model, or in an unadjusted analysis, and was dropped from the regression model. Age was not a statistically significant predictor (p = 0.08), but was included in the final model, as it might be an important confounder. SES was included as a linear term in the model. An interaction between urban/rural location and SES was included in the final regression model, to examine effect modification of the relationship between SES and availability of UDM first grade flour by location. The conditional odds ratios from this regression model were examined to help understand predictors of using UDM flour.The strongest predictor of having UDM first grade flour was whether or not a woman bakes her own bread . Knowledge that UDM flour is fortified was associated with an increased likelihood of having the flour in the household . Women with a history of anemia were slightly less likely to have first grade UDM flour in the house than those with no history , though this result was not statistically significant. Women who had taken iron supplements within the last two years  and those who had taken iron tablets more than two years ago  were slightly more likely to have UDM first grade flour in the household than those who had never taken iron, but this result was also not statistically significant (p = 0.23).Among rural women, there was no strong evidence of an association between SES and having flour in the household . Among urban women, decreasing SES was associated with an increase in UDM first grade flour possession .There were no statistically significant differences in anemia prevalence or iron depletion prevalence by possession of UDM flour  or possession of grey loaf .Those who had a grey loaf in the household the day before the survey had a significantly lower (p = 0.03) prevalence of folate deficiency, with an estimated prevalence 22.7%  among those with grey loaf compared to 29.7%  among those without the grey loaf. Those who had UDM flour in the household had a higher prevalence of folate deficiency (p = 0.03), with an estimated prevalence of 26.2%  among those without UDM flour compared to 30.8%  among those with UDM flour. However, UDM and grey loaf possession are both highly correlated with urban/rural location and socioeconomic status; the associations between UDM/grey loaf possession and folate deficiency were not statistically significant in a multivariate logistic regression analysis adjusting for SES or urban/rural location. Consequently, the decrease in low folate prevalence should not be directly attributed to grey loaf possession or the increase in low folate prevalence to possession of UDM flourAmong WRA in Uzbekistan, 54.5%  reported that they had heard of fortified food. Nationally, 47.0%  of respondents mentioned first grade UDM flour or the grey loaf among the types of fortified food. Among WRA, 32.1%  were aware that first grade UDM flour or the grey loaf are fortified with iron. However, only 3.7%  of respondents were able to mention the benefits of fortified flour/bread consumption, and only 12.2%  of respondents were aware how to distinguish fortified flour from non-fortified flour. The “non-polvon” logo  for fortification was recognized by 36.9%  of women; however, only 23.1%  of respondents understood the meaning of this healthy food logo. The level of knowledge of the women about IDA was low, as only 12.5%  of women were able to identify IDA causes  and 17.4%  knew who the most-at-risk populations were. Only 41.9%  of WRA were able to list at least three signs or consequences of anemia, and 5.9%  of the women were aware how to avoid IDA.The estimated proportion of WRA in Uzbekistan who ate heme iron-rich foods  at least twice in the last week was 96.2% ; and at least once the day before the survey was 91.4%; . Consumption of enhancers of iron absorption  was also high, with 90.2%  consuming these foods at least twice in the week prior to the survey; and 97.1%  consuming these foods the day before the survey. However, consumption of iron absorption inhibitors during a meal or directly after a meal was also high, with 94.7%  of women reporting drinking coffee or tea with a meal the day before the survey.To assess the impact of dietary choices on the biochemical measures, the consumption of iron-rich foods at least twice in the week preceding the survey was used as a proxy for consumption of heme iron containing foods in general, as this indicator should reflect regular consumption of heme rich foods. There was no evidence of an association between consumption of heme rich foods and iron depletion (p = 0.29) or anemia (p = 0.68); only 97 women (3.8%) did not regularly consume heme rich foods. The estimated prevalence of low ferritin in those who ate ‘heme iron-rich foods’ foods was 47.8%  compared to 41.4%  in those who did not; and the prevalence of anemia was 34.4%  in those who ate the foods versus 32.1%  in those who did not eat the foods.There was marginal evidence of an association between low folate concentrations and consuming heme iron-rich foods (p = 0.07); the prevalence of low folate concentrations in those who ate heme iron-rich foods was 28.5% , compared to 39.1%  in those who did not. When adjusted for relevant confounders  in a multivariate analysis, this association was attenuated and not statistically significant (p = 0.18).In this paper, coverage of the flour fortification program and biochemical measures of folate deficiency, iron deficiency, and anemia among WRA are described following the implementation of a national flour fortification program. By measuring the biochemical measures, alongside coverage of fortified products and knowledge of dietary and health concepts, areas with potential for improvement were identified. The LC-LQAS survey was carried out three years after the initiation of the NFFP, in order to give sufficient time for the fortification program to have an impact on the target population group, WRA.The aim of reducing the prevalence of anemia in non-pregnant WRA to 48% was surpassed, as anemia prevalence was estimated at 34.4% . Although evaluating the direct impact of the fortification program is difficult, it is likely that the UDM first grade flour and grey loaf fortification project contributed to this decline in anemia prevalence. However, the interpretation of the biochemical results might also be somewhat compromised, as the survey coincided with the time the premix was in short supply.The national survey results were compared to the sentinel-site data based on 40 households collected between 2004 and 2007. Results from the sentinel study should be interpreted with caution, as they were not nationally representative. Nevertheless, between 2004 and 2007, estimates of anemia prevalence decreased by 23.5% (from 51.3% to 27.8%), the 2007 result being comparable to that obtained in the LC-LQAS survey (34.4%) reported here. Confidence intervals for the sentinel study were not available, but the decline was not statistically significant. The sentinel site study and the LC-LQAS survey cover the time period of the NFFP and suggest a positive effect of the fortification program on anemia prevalence.The estimated overall prevalence of depleted iron stores (ferritin<15 µg/L) increased from 28.2% in 2004 to 33.3% in 2007 in the sentinel study and was highest in the LC-LQAS survey (47.5%). The higher prevalence of depleted iron stores in the LC-LQAS survey was likely due to the inclusion of a representative sample from all regions of the country, including the poorer regions. In the Republic of Karakalpakstan, the prevalence of low ferritin was the highest in the country (63.9%). Sub-clinical inflammation, indicated by elevated concentrations of the acute phase proteins, could impact on serum ferritin concentrations causing them to increase, thus underestimating depleted iron stores In Uzbekistan, the UDM first grade flour is often mixed with other flours during bread making, diluting the fortification effect; a more bioavailable form of iron might help improve the biological impact. Later versions of the KAP premix have used more bioavailable sources of iron, such as NaFeEDTA or ferrous sulfate and are an option, which could be considered.In the LC-LQAS survey, an estimated 29.1% of women were folate deficient, similar to the prevalence obtained in 2004 (33.3%) and 2007 (24.1%) from the sentinel study. Folate deficiency among WRA is likely driven by low folate intake in the diet or cooking processes that destroy folate. Consistent consumption of adequately fortified flour should improve folate concentrations in WRA. The prevalence of folate deficiency varied significantly by oblast and may be explained by an iron-folic acid supplementation program, distributing weekly tablets to WRA in some oblasts at the time of the survey; however, there is no data on which oblasts continued to get the supplements.Folate and iron deficiency were strongly associated with stage of anemia. While women with both iron and folate deficiency had the most severe anemia, folate deficiency was associated with more severe anemia than ferritin deficiency, suggesting that folate was a limiting nutrient in iron nutrition. Folate is needed for the formation of hemoglobin, and deficiency impairs the maturation of young red blood cells, resulting in anemia Overall, the flour fortification program was implemented successfully, with fortified flour available throughout Uzbekistan. At the time of the survey, 59.8%  of households reported having UDM first grade wheat flour or bread made from the flour. The fortified grey loaf was more popular than UDM flour in the urban locations of Tashkent City and Tashkent Oblast where there are more bakeries selling ready-made bread. Combining the data together, 71.3% of households  had either a grey loaf or home-baked bread made from UDM first grade flour on the day of the survey, which is close to the program target of 80% of households.UDM first grade flour coverage varied geographically and was highest among low SES and rural women. This flour is perceived as being of poor quality, and those who can afford the more expensive non-fortified Kazakh flour will purchase it. Conversely, in areas where there are higher rates of poverty, such as the semi-autonomous Republic of Karakalpakstan, households largely consume flour that they produce and mill themselves (non-fortified); rates of anemia and depleted iron stores were higher among women in these regions relative to the rest of Uzbekistan. No statistically significant associations were observed between anemia or iron depletion prevalence and possession of fortified products. Associations between possession of fortified products and folate deficiency may be attributable to socioeconomic and location differences between those with and without the fortified products.In early 2008, there was a sharp drop in fortified first grade flour milled due to a shortage of fortificant. Premix supplies were available again in June 2008, but it was not until later in 2008 that production once again reached >90% of total UDM first grade flour milled . The iron spot test found 41.6%  of the flour was adequately fortified, and the fortification program target of 30% non-pregnant WRA regularly consuming fortified wheat flour was achieved. However, there was substantial variability by oblast in the percentage of adequately fortified flour samples. While UDM has flour-mills in all oblasts, the availability of premix, as well as the quality of the fortification process, may vary across mills. An independent end-of-project evaluation was carried out in 2009, subsequent to the survey and the premix shortage. This evaluation reported that 46% of iron spot tests at mills showed adequate fortification Knowledge about fortified foods and the fortification project was low among women. While the healthy food logo was recognized by 36.9% of WRA, only 23.1% of women understood what the logo represented and only 12.5% of WRA knew how to distinguish fortified flour from non-fortified flour. Measuring whether target groups had seen the logo says little about how WRA link this logo to fortified food or how the logo might affect food purchasing or consumption habits. The success of the fortification program requires that women understand and accept that fortified flour improves their health. The survey results suggest that communication messages for fortification might need to move from logo recognition to include a larger component of consumer nutrition education.Less than half of the women interviewed knew the signs and consequences of IDA, and even fewer knew the causes of IDA. As with the logo recognition, more widespread health education is needed at opportune times, e.g. such as visiting SVP/policlinics or in schools for girls and distributing nutritional information to both women and girls.The diet of the women consisted of foods that can reduce the risk of anemia, including iron-rich foods, such as meat, fish or poultry, and iron-enhancers such as vegetables and fruits. Data on food frequency from the 2002 Health and Examination Survey suggest that red meat was eaten by women on average 5 times per week, but rural (3.9 days)/urban (7 days) differences existed The fortification of first grade UDM wheat flour with a variety of micronutrients, including iron and folic acid, coincided with a substantial decline in the prevalence of anemia in WRA in Uzbekistan. But the high prevalence of depleted iron stores suggests that women are still not eating enough iron in the diet or are drinking too much tea at meal times and inhibiting iron absorption. However, prioritizing folate over iron intake may have more of an impact on anemia prevalence in this population.The government now recommends intake of a weekly folic acid and iron tablet for WRA, children and adolescents to ensure an adequate supply of folate and iron. For non-pregnant women and adolescents, the tablet contains 350–500 µg of folic acid and 60 mg iron; and for pregnant women, 700–1000 µg folic acid and 120 mg iron. Infants <1 year are given a smaller dose of iron (30 mg). This governmental action should help decrease the prevalence of observed folate and ferritin deficiencies, and subsequently anemia.The data from the 2008 LC-LQAS survey showed that use of fortified flour was common throughout the country. However, a higher proportion of UDM flour needed to be fortified to the appropriate level and fortification levels adequately monitored. To address this issue, mandatory fortification legislation was enacted in January 2011; this legislation stipulated that mills, both state-run and private, must meet fortification standards set for first grade flour in order to receive annual production permits. Further, in June 2011, a Presidential decree stipulated the specific vitamin-mineral mixture for the enrichment of bread making wheat flour, and stated that the flour must contain 1 mg folic acid and 33.3 g iron/100 g wheat flour (O'z DSt1098: 2011). Additionally, the decree stipulates that 1.3 mg thiamin, 2.0 mg riboflavin, 6.7 mg niacin and 14.7 mg zinc/100 g wheat flour should be added. Once flour is consistently fortified at an adequate level, this decree should also help address the observed folate and ferritin deficiencies.To address the need for better monitoring of the program, the two laboratories responsible for external quality assurance of the fortified flour have been provided with all equipment and training needed to ensure the quality control of the fortification process. However, unfortified flour from Central Asia continues to be imported, diluting the fortification effect. A regional approach to domestic and imported flour legislation, coupled with common standards on fortification dosage and quality, would likely achieve the coverage of fortified flour consumption to produce sustainable health improvements.The data from the survey suggested communication messages about the fortification project were not reaching the majority of women, and women's understanding of the causes of IDA was poor. Since 2008, customization of social marketing activities targeted at different population groups, e.g. consumers, producers (millers and bakers) and the media, have been implemented as an innovative and effective approach. For example, the packaging standards mandate that producers print the logo “Enriched with Iron” on flour packages and education of millers and bakers on the health implications of micronutrient deficiencies have been used to build awareness.


N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs) or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-)activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced  Arabidopsis thaliana belongs to an intriguing class of papain-like Cys proteases (PLCPs), typified by the presence of a C-terminal granulin domain rd21 lines have enhanced susceptibility for the necrotrophic pathogen Botrytis cinereaRD21 ortholog in Nicotiana benthamiana renders the plant susceptible for the oomycete pathogen Phytophthora infestansRD21  of Relatively little is known about the regulation of RD21 activity. Arabidopsis RD21 is composed of five domains: a signal peptide; an autoinhibitory prodomain; the protease domain; a proline-rich domain; and a granulin domain (Arabidopsis RD21 is present in the vacuole and in Endoplasmic Reticulum (ER)-bodies RD21 activity has been detected in Arabidopsis leaf extracts using protease activity profiling Emerging roles of RD21-like proteases in immunity, and their association with various types of stress, prompted us to subject RD21 to further biochemical characterization. In this study, we addressed a series of questions regarding location, activation, maturation and regulation of (mutant) RD21 upon expression in plants. These studies show that RD21 traffics through the Golgi and reveal three consecutive layers of post-translational regulation of RD21. The studies also illustrate the use of agroinfiltration as a protein production platform, and the reliability of DCG-04 profiling as a tool to detect active protease isoforms.N. benthamiana in the presence of silencing inhibitor p19 to boost the expression levels We transiently overexpressed RD21 by agroinfiltration of leaves of N. benthamiana (see below).The anti-RD21 protein blot shows strong signals upon RD21 overexpression, when compared to the empty vector (EV) control . The 25 kDa signal (band 3) not only contained peptides of the protease domain of RD21, but also peptides from an aleurain-like protease of N. benthamiana . The presence of NbALP in the 25-kDa band indicates that the biotinylated signal in the empty vector control is also caused by NbALP . No tryptic peptides of the signal peptide, prodomain or granulin domain were found, consistent with the absence of these domains in mRD21. Furthermore, the peptide carrying the catalytic Cys was also not detected. This catalytic Cys resides in a 60-residue tryptic peptide (MW 6500 Da), which is too large to be detected, especially when biotinylated.To confirm the identity of the proteins labeled by DCG-04, biotinylated proteins were purified on streptavidin beads, separated on protein gels and analyzed by mass spectrometry  in the prodomain, and one in the granulin domain , Picea stichensis (gi|116786779) and Pseudotsuga mendiesii (gi|1208549). Importantly, western blot analysis show that all isoforms of the +PGS RD21 mutant are slightly larger than the corresponding isoforms of wild-type RD21, consistent with N-glycosylation of the protease domain , and a mutant in which three adjacent Cys residues in the granulin domain are replaced by Ala residues (3C3A). The latter mutant is aimed at destabilizing the granulin domain, since these three Cys residues are presumably involved in three disulphide bridges that stabilize the granulin domain N. benthamiana. Furthermore, despite the absence of the catalytic Cys, a weak, E-64-competable biotinylation occurs of iRD21  plants, representing mRD21 and iRD21, respectively , intermediate isoform (iRD21) and three isoforms of mature RD21 (mRD21). ProRD21 is normally not found in leaf extracts, but this precursor becomes detectable upon extraction in the presence of E-64, suggesting that some proRD21 accumulates in agroinfiltrated leaves and that residual prodomain removal occurs during extraction. However, RD21 in leaf extracts is mostly a mixture of intermediate and mature isoforms. The intermediate iRD21 form aggregates and precipitates during purification Prodomain removal was blocked in the ΔPG deletion mutant, but not in the ΔG deletion mutant. This indicates that the proline-rich domain is required for prodomain removal. However, the presence of a 25 kDa mRD21 isoform suggests that the proline-rich domain is not necessary for protease activity once the prodomain is removed. The granulin domain is not required for prodomain removal and folding since both the ΔG deletion and 3C3A destabilization mutants accumulate as active mRD21.N-glycosylation site (PGS) in the prodomain and another one in the granulin domain, mutagenesis and PNGaseF treatment demonstrated that the PGS in the granulin domain is probably not N-glycosylated, possibly because it is buried in the granulin domain and inaccessible for N-glycosylation enzymes in the ER N-glycosylation status of the prodomain has not been addressed in this study.Although RD21 contains a putative N-glycosylated, evident from an increased molecular weight of all RD21 isoforms. Deglycosylation experiments with PNGaseF indicated that the N-glycan is α-1,3-fucosylated, a hypothesis that was confirmed by transient expression in N. benthamiana plants silenced for the respective α-1,3-fucosyltransferase. Fucosylation of glycoproteins is common in plants but does not always occur at all possible sites. N-glycosylation of the membrane-resident receptor-like protein Cf-9, for example, occurs at 21 of the 22 PGSs, but all sites are PNGaseF sensitive, indicating that none of these N-glycans are fucosylated N-glycosylation of two sites on phytohemagglutinin in Phaseolus vulgaris: one was fucosylated and the other was not. These data illustrate that fucosylation depends on the accessibility of the N-glycan on the folded protein.We introduced a PGS in the protease domain at a position where PGSs occur in other RD21-like proteases. This mutant RD21 becomes indeed N-glycan can not be removed by EndoH, which can only remove oligomannosidic  N-glycans. Furthermore, the N-glycan of RD21 is fucosylated, which requires a fucosyltransferase activity that resides in the medial Golgi RD21 is known to accumulate in the vacuole in planta. The presence of some proRD21 signal in the C161A mutant may be caused by trapping of proRD21 in the large amount of iRD21 aggregates. A non-autocatalytic prodomain removal is unusual since many PLCPs remove their own prodomain during acidification of their environment, e.g. in the lysosome, the lytic vacuole or the apoplast The majority of the C161A mutant accumulates as iRD21 even though this mutant is inactive in rubisco degradation and DCG-04 labeling. This observation implies that the catalytic Cys is not required for prodomain removal vpe quadruple knockout lines  interacts and colocalizes with RD21 In conclusion, we have studied three levels of post-translational regulation of RD21 activity. The first level is the removal of the prodomain. Prodomain removal is essential for activation of RD21 and is not autocatalytic and presumably involves another protease. The second level is the conversion of iRD21 into mRD21 by removal of the granulin domain. This maturation is probably autocatalytic since it requires the catalytic Cys and His residues. This maturation is thought to increase the proteolytic activity since iRD21 resides in aggregates and will have less access to substrates, whereas mRD21 is a soluble protease rd21-1 knockout lines, illustrating that RD21 is a major proteolytic activity affecting proteome stability in leaf extracts.The activation of latent RD21 by SDS may also have important practical implications. SDS and DTT are standard components of SDS-PAGE loading buffers and often thought to denature and inactivate proteolytic activities. Our experiments illustrate that these conditions rather activate RD21, causing slow, but significant proteome degradation. Both SDS and DTT are required for this process and proteomes are more stable in extracts from Our studies show a consistent correlation between DCG-04 labeling and protein degradation. For example, the catalytic C161A and H297A mutants, and the ΔPG deletion mutants are unable to degrade rubisco and are poorly labelled with DCG-04. The catalytic N317A mutant is slow in rubisco degradation and DCG04 labeling when compared to wild-type RD21. Furthermore, SDS-induced rubisco degradation is associated with increased DCG-04 labeling in Arabidopsis leaf extracts. These observations support the notion that protease activity profiling is a reliable prediction of protease activity.N. benthamiana by agroinfiltration for medical purposes has become commercially attractive N. benthamiana to very high levels. The proteins pass through the Golgi, receive the necessary post-translational modifications and accumulate presumably in lytic vacuoles to relatively high concentrations that nevertheless remain harmless for the plant.Besides structure-function analysis of RD21, our studies also provide useful observations for agroinfiltration as protein production platform. Production of heterologous proteins in Tables S1 and S2. The following plasmids have been described previously: pRH80 Table S2. The verified mutant expression cassettes were shuttled into binary vectors as summarized in Table S1, resulting in a collection of binary plasmids (Table S3) that all carry a T-DNA with a 35S-driven (mutant) RD21 open reading frame, followed by a terminator of the potato PI-II gene.A summary of the cloning is provided in Agrobacterium tumefaciens GV3501 pMP90 2, containing 0.2 µM acetosyringone. Bacteria were diluted to OD600 = 2 and mixed with an equal volume of Agrobacterium carrying a binary plasmid that encodes silencing inhibitor p19 600 = 0.4. The mixed cultures were kept at room temperature for 1 hr and infiltrated into the two youngest, fully unfolded leaves of 4-week old N. benthamiana plants.The binary vectors were transformed into N. benthamiana leaves at the third day after agroinfiltration were cut into small pieces with a razor blade and ground in 10 mM Tris pH8, containing 5 mM DTT, in an ice-cold mortar. After removing insoluble materials by repeated centrifugation at 20,000 g for 5 min at 4°C, the protein extracts were adjusted to a concentration of 1 mg/mL. 100 µg of protein extracts was labeled in 500 µL 100 mM sodium acetate pH 6, containing 1 mM DTT, with 0.2 µM DCG-04 for 1 hr at room temperature. In no-probe-controls (NPC) 1 µL DMSO was added instead of 1 µL 1 mM DCG-04 in DMSO. In E-64 treatments, an extra 20 µM E-64 was added before adding the probe. The labeling reactions were stopped by adding 1 mL of 100% acetone at −20°C. The proteins were pelleted by centrifugation at 20,000 g for 5 min at 4°C and the protein pellets were dissolved in 50 µL SDS-PAGE loading buffer containing β-mercaptoethanol. The proteins were separated on 12% SDS PAGE gels (10 µg protein per lane) and the detection of biotinylated proteins was done as previously described using streptavidin-HRP (Sigma-Aldrich) Leaf discs without main veins of agroinfiltrated N. benthamiana (NbWT) and transgenic N. benthamiana silenced for both β-1,2-xylosyltransferase and α-1,3-fucosyltransferase  Sequences of Arabidopsis RD21 and N. benthamiana ALP . The signal peptide and prodomain are indicated in light and dark grey, respectively. The identified tryptic peptides and catalytic residues are underlined and boldface, respectively. B) Summary of the identified tryptic peptides, with individual scores. *, oxidized methionine; **, X correlation value: >1.5 maybe; >2.5 good; >3.5 excellent; >4.5 no doubt. Only tryptic peptides are shown.(PDF)Click here for additional data file.Table S1Cloning details.(PDF)Click here for additional data file.Table S2Primer sequences.(PDF)Click here for additional data file.Table S3List of binary plasmids used in this work.(PDF)Click here for additional data file.


In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors , to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving  From these basic building blocks, enzymes have evolved to perform the vast repertoire of chemical reactions, many of which are highly complex, found in nature. They do this under physiological conditions  with phenomenal yields and exquisite stereoselectivity and regioselectivity, a continuing goal for many synthetic organic chemical processes, which often need very harsh conditions to perform the same chemistry. Enzymes are also responsible for the uptake, synthesis and breakdown of chemicals, such as drugs or environmental contaminants (e.g. pesticides), in our bodies. In humans, thousands of enzymes control the rates of essential cellular reactions, and enzymes represent ∼ 63% of all drug targets  .As has been recently shown , there ain vivo; thus, the biological unit must be derived from the data in the Protein Data Bank (PDB) and associated biochemical information.An enzyme is composed not only of the catalytic components (residues and cofactors) but also of many amino acids that make up the protein’s functional biological unit. It is worth noting that, in protein crystallography, the structure is determined in a crystalline environment, which may not reflect the functional biological unit EC 1.11.1.10). There are three different types of this enzyme that have been identified so far, all of which catalyse the same basic reaction  to specific proteins, as defined by their sequences and structures. This mapping is complex and is rarely 1 : 1. Some enzymes perform multiple reactions; some reactions are performed by multiple unrelated enzymes. In addition, different enzymes with an identical EC number  occasionally have significantly different mechanisms. A good example of this phenomenon is provided by the haloperoxidases  and structures (for a lower limit). Although this approach does not take into account that different EC nodes, especially within a sub-subclass, may have very similar mechanisms and relatives with respect to their evolution, the fact that they have different substrate specificities or different reaction outcomes (products) is considered to be important in this analysis. It is further worth noting that, as not all EC nodes have associated biological data , the following can only be considered to be rough estimate.In order to gain a better idea of the scope of this complexity and determine the total number of reactions It has previously been suggested that, whereas the theoretical limit on the size of protein space is astronomically large , it is entirely feasible that most  of functionally relevant protein sequence space has already been explored . Furtherblastclust (part of the NCBI BLAST package), and identified a total of 13 150 sequence clusters, suggesting that there are, on average, 4.95 distinct evolutionary families per EC node. However, blastclust produces many singleton ‘clusters’, so the use of hidden Markov models allows many of these singleton clusters to be placed with other clusters. This gives a total of 9312 clusters, and an average of 3.50 distinct evolutionary families per EC node. That is, there are, on average, a little over three different ways in which the same overall chemistry can be performed. Almost 50% of EC nodes are only represented by a single evolutionary family (i.e. a single cluster), but this may be attributable to a lack of assigned sequences in the databases. The other half of the EC nodes all contain more than one family, with the nonspecific Ser/Thr protein kinase (EC 2.7.11.1) being the most promiscuous EC node, with 450 associated sequence families.We identified 2657 EC nodes (a fully defined EC number) that also had a sequence assigned . For each of these EC nodes, we clustered the sequences using In order to compare these results more accurately with the structure analysis which follows, we can calculate the number of families per EC node for which both a sequence and a structure have been identified. Given that only approximately one-quarter of all EC numbers actually have a solved crystal structure deposited in the PDB, the number of families drastically decreases to an average of 2.02 evolutionary families per EC node.An enzyme is not just a sequence or an active site; it is a complete protein that can be composed of many different chains and structural domains. We can define a lower limit to the number of times that a reaction has evolved by utilizing the principle that, during protein evolution, two proteins may have significantly diverged in terms of sequence similarity, but may still have a common ancestor (and thus function), which can be identified through structural similarity, as this is maintained for much longer than sequence similarity .Protein structure can be categorized in many different ways, but the two most common are CATH   and SCOPEC 2.7.11.1) reaction, with 58 evolutionary families at the structural level. It is unclear at this time exactly why the nonspecific Ser/Thr protein kinases are so diverse in terms of their protein families.By taking the unique combinations of CATH domains and incorporating sequence information see , we obtaAlthough there are many EC nodes yet to be fully assigned in terms of both sequence and structure, we do not expect that the average number of families per EC node will change significantly, because, between 2009 and 2011, the average number of families per node changed by a value of 0.01, and almost 50 000 enzyme sequences were added to Swiss-Prot.Although biological activity requires the complete biological unit, not all of the protein structure may be catalytic, and so we can identify certain domains as catalytic and others as binding or noncatalytic. A catalytic domain can be defined as any CATH domain that furnishes at least one catalytic residue. An analysis of proteins annotated as enzymes in version 3.3 of CATH identified 1077 domains unique at the H level, of which only 411 distinct domains were catalytic in both MACiE and the CSA .These domains can be further linked with the annotations of EC number through their assignment to specific PDB codes (which are assigned EC numbers). Although over one-quarter of the catalytic CATH codes are only represented by a single EC number , there are some domains that are incredibly promiscuous with respect to the number of overall reactions in which they are involved . The mosEC 1.2.99.2, M0107), and the largest catalytic unit that we have identified to date contains 15 domains, two of which are involved in forming the active site .Whereas the biological unit is the assembly of domains and chains that is found in the cell and is required for full biological activity, we can further define a catalytic unit as the smallest assembly that is conceptually required for catalytic activity, i.e. the single unit containing the active site. Thus, a biological unit may have several catalytic units (and thus active sites). In the MACiE database, we only annotate a single catalytic unit, so we manually examined MACiE to identify the domain composition of the catalytic unit for each protein . The different domain compositions of the enzymes in MACiE can be represented as shown in More generally, only one-third of enzymes in MACiE are truly single-domain proteins in that they have a single domain in both their catalytic and biological assemblies. However, these single-domain enzymes span all EC classes of chemical reactions, although there does appear to be a slight preference for the hydrolases (EC 3) to be single-domain proteins . Whilst EC 1.2.4.1, M0106), in which the catalytic site lies at the interface between two different chains, and although the catalytic domain is the same in each chain (CATH 3.40.50.970), one of the chains also has a second domain, which is not part of the active site. Thus, it can be seen that an enzyme is often composed of more than simply the active site or just the catalytic domains. Finally, it is worth noting that an enzyme can also have multiple active sites. For example, in glutamine-fructose-6-phosphate transaminase , the first part of the reaction (the conversion of l-glutamine to l-glutamate and ammonia) is performed in one domain (CATH 3.60.20.10 on chain A) and the ammonia is then transferred through a channel in the protein to the second active site, where it is used in the second half-reaction (the conversion of d-fructose 6-phosphate to d-glucosamine 6-phosphate), which occurs at the interface between chains A and B, both of which have the same CATH code (3.40.50.10490).A further complexity lies in the fact that an active site can be located in a single domain, at the interface between two domains, or even on multiple chains. Other arrangements include cases such as pyruvate dehydrogenase  between different substrates ; and (b) the domain combination of the enzymes may also change, and this may lead to changes in function  – a good example of the domain combination leading to different functions is provided by the Ntn-type amide hydrolase enzymes, which all have the CATH domain 3.60.20.10 in common; however, combination of this domain with a second, distinct domain changes the function of the enzyme, sometimes rather dramatically.Although we have investigated the number of times that a specific function as defined by the EC node has evolved, we have not looked in detail at the evolution of function in general. Our own observations, along with work from the Babbitt laboratory , have deAlthough many such changes can be identified with the use of MACiE, they are beyond the scope of this review, which treats all EC nodes in isolation from one another.The fact that, on average, an overall function appears to have evolved twice might be further evidence of divergence of function, in that similar catalytic apparatus can perform similar but distinct functions when presented with new situations, or indeed that domain combinations can be changed to change function. However, there are still many questions that are not fully answered, and this can only be achieved by combining sequence and structure with the chemistry, mechanism and phylogeny.Over time, enzymes have evolved to be fit for purpose, and we see how nature has, on occasion, used multiple mechanisms to perform the same reaction or evolved a single protein family to perform many different reactions. This offers an insight into how we might proceed towards designing enzymes with novel functions, or how we might modulate the functions of known enzymes to perform specific tasks. However, we still need better tools with which to characterize and compare enzyme reactions, and we need better methods with which to elucidate and validate mechanisms. The MACiE database provides the basic data from which we can begin to obtain a better understanding of mechanisms and with which we will hopefully be able to predict and even design new mechanisms, given an enzyme sequence or structure.


The major role of enzymatic toxins that target nucleic acids in biological conflicts at all levels has become increasingly apparent thanks in large part to the advances of comparative genomics. Typically, toxins evolve rapidly hampering the identification of these proteins by sequence analysis. Here we analyze an unexpectedly widespread superfamily of toxin domains most of which possess RNase activity.The HEPN superfamily is comprised of all α-helical domains that were first identified as being associated with DNA polymerase β-type nucleotidyltransferases in prokaryotes and animal Sacsin proteins. Using sensitive sequence and structure comparison methods, we vastly extend the HEPN superfamily by identifying numerous novel families and by detecting diverged HEPN domains in several known protein families. The new HEPN families include the RNase LS and LsoA catalytic domains, KEN domains (e.g. RNaseL and Ire1) and the RNase domains of RloC and PrrC. The majority of HEPN domains contain conserved motifs that constitute a metal-independent endoRNase active site. Some HEPN domains lacking this motif probably function as non-catalytic RNA-binding domains, such as in the case of the mannitol repressor MtlR. Our analysis shows that HEPN domains function as toxins that are shared by numerous systems implicated in intra-genomic, inter-genomic and intra-organismal conflicts across the three domains of cellular life. In prokaryotes HEPN domains are essential components of numerous toxin-antitoxin (TA) and abortive infection (Abi) systems and in addition are tightly associated with many restriction-modification (R-M) and CRISPR-Cas systems, and occasionally with other defense systems such as Pgl and Ter. We present evidence of multiple modes of action of HEPN domains in these systems, which include direct attack on viral RNAs (e.g. LsoA and RNase LS) in conjunction with other RNase domains , suicidal or dormancy-inducing attack on self RNAs (RM systems and possibly CRISPR-Cas systems), and suicidal attack coupled with direct interaction with phage components (Abi systems). These findings are compatible with the hypothesis on coupling of pathogen-targeting (immunity) and self-directed (programmed cell death and dormancy induction) responses in the evolution of robust antiviral strategies. We propose that altruistic cell suicide mediated by HEPN domains and other functionally similar RNases was essential for the evolution of kin and group selection and cell cooperation. HEPN domains were repeatedly acquired by eukaryotes and incorporated into several core functions such as endonucleolytic processing of the 5.8S-25S/28S rRNA precursor (Las1), a novel ER membrane-associated RNA degradation system (C6orf70), sensing of unprocessed transcripts at the nuclear periphery (Swt1). Multiple lines of evidence suggest that, similar to prokaryotes, HEPN proteins were recruited to antiviral, antitransposon, apoptotic systems or RNA-level response to unfolded proteins (Sacsin and KEN domains) in several groups of eukaryotes.Extensive sequence and structure comparisons reveal unexpectedly broad presence of the HEPN domain in an enormous variety of defense and stress response systems across the tree of life. In addition, HEPN domains have been recruited to perform essential functions, in particular in eukaryotic rRNA processing. These findings are expected to stimulate experiments that could shed light on diverse cellular processes across the three domains of life.This article was reviewed by Martijn Huynen, Igor Zhulin and Nick Grishin Over the past decade it has become increasingly evident that the deployment of enzymatic toxins that target nucleic acids is a common feature of biological conflicts at all levels -5. TheseAnother common class of inter-genomic conflicts is that between viruses and the host cell genome . In thesRecent studies indicate that many of the RNases related to those that participate in biological conflicts are involved in core cellular functions as RNA-processing enzymes. A case in point is the EndoU RNase domain that apparently was derived from ancestral RNases found in polymorphic toxins and related systems . Upon acOur previous work has shown that investigation of proteinaceous toxins using sensitive sequence analysis and structure comparison techniques, combined with contextual information derived from genome comparisons, has considerable potential for discovery of new RNA-targeting activities ,10,35. HWe describe here comprehensive sequence, structure and genomic context analyses that strongly support the interaction of the HEPN domain with nucleic acids in multiple systems involved in biological conflicts and processing of cellular RNAs. In particular, we present evidence that several diverse HEPN versions function as metal-independent RNases. Thus, the RNase activity of HEPN domain could be a unifying theme shared by cellular RNA maturation systems and those involved in biological conflicts.Transitive, iterative sequence profile searches and hidden Markov model (HMM) searches with the originally defined HEPN domains ,42 used The search strategy outlined above identified numerous families of domains  we observed that the H is typically absent in the Rx4-6H motif though the basic residue corresponding to the R is present. However, these proteins have a second strongly conserved H occurring N-terminal to the above motif. Examination of the structure reveals that this H could constitute an alternative active site similar to the classical HEPN active along the basic residue from the motif  in the Parep1/8 family of the HEPN-T clade , we retSulfolobus solfataricus (PDB: 2i71) and Pyrococcus furiosus (PDB: 4eog) .-92.Brachs Figure . Given tHaloplasma contractile; Figure E. coli RNase LS and LsoA is interrupted by a stop codon, leaving HEPN as the only active nuclease domain. The presence of this RNase H module   catalytically active HEPN domains and other active RNase domains resulting in “two-headed RNases”. A case in point is the fusion of HEPN with a C-terminal RNase III and a dsRBD domain , represent anti-bacteriophage strategies that limit the spread of infection [Lactococcus lactis AbiTii protein lacks a HEPN domain, its homologs from several bacteria are found fused to two distinct C-terminal HEPN domains namely of Ymh (gi: 372210551) and the c2405 (gi: 358446093) families. The mode of action of these Abi proteins has remained largely enigmatic to date. The detection of HEPN domains suggests a unified mechanism for their action, based on the predicted RNase activity. For example, AbiD1 has been shown to be toxic to the host cell but also to interfere with the activity of the RuvC-like Holiday junction resolvase of phage bIL66 [L. lactis AbiD1 induces cell death at suboptimal temperatures and is also toxic in heterologous systems such as E. coli[The abortive infection (Abi) systems, of which over 22 distinct versions were first characterized in nfection . Homolognfection . They apnfection ,103. Thuge bIL66 ,101. Furs E. coli,105. Alts E. coli. Taken tThe recognition of this general mode of action for the HEPN-containing Abi systems also leads to a hypothesis on the functions of 5 novel protein domains that we detected at the N-termini of different groups of proteins of the AbiJ family and the conserved domain in AbiTii Table  and 4. WThus, there is a strong tendency toward multiple, independent fusions of these NTDs with both RNases and DNases, as well as other catalytic domains such as protein kinases that might also function as toxins Figure . AlthougComparable to the Abi systems, HEPN domain proteins are also major players in Type I and Type III CRISPR-Cas adaptive immunity systems in archaea and bacteria . There aE. coli (Table Neisseria and Kingella). These ABC-HEPN proteins might perform roles similar to those proposed for the CARF-HEPN proteins.Beyond the PrrC-like and RloC-like families of HEPN proteins, we detected several additional fusions of ABC-ATPases with C-terminal HEPN domains, e.g. those prototyped by the APECO1_4465 (gi: 117623091) protein from avian pathogenic Streptomyces coelicolor  system that was first characterized as a defense system against lysogenic phages (e.g. phiC31) in r Figure . The Pglr Figure  [102,116Legionella pneumophila, that is secreted through a type-IV secretion system and contains a nucleotidyltransferase-HEPN fusion, functions as a toxin that targets eukaryotic host cells [The organization of genetic elements which encode the originally identified HEPN domains and MNTs clearly resembles Type II TAs suggesting that these elements are novel TA systems  althoughst cells . The nucst cells , suggestst cells . In sevest cells ,7,10,35.st cells . This inst cells , a potenst cells . Under tst cells .We also uncovered a similar but less common gene dyad that combines a HEPN gene of the MAE_18760 family Table  with a gA further wrinkle regarding the MNT-HEPN systems relates to the mode of action of the HEPN toxins. A subset of the HEPN domains found in these systems preserve the Rx4-6H motif (in large part represented by the DUF86 family in Pfam) or have alternative histidines and and are likely to function as endoRNases, similar to toxins in various TA systems. However, no conserved, potential active site residues are found in several HEPN domains from the MNT-HEPN systems  that are associated with certain mobile elements, such as integrons, which are major vehicles in the spread of drug resistance determinants among proteobacterial pathogens . The intVibrio cholerae) and contains a distinctive N-terminal domain with a single TM helix with a strictly conserved WP signature . In the third group the N-terminal HEPN domain is separated from the C-terminal Zn-ribbon domain by a pair of TM helices (e.g. gi: 325108440 from Planctomyces brasiliensis). All these HEPN domains are predicted to be the cytoplasmic globular domains of inner membrane proteins. This localization suggests that, similar to the Ire1 and C6orf70 proteins in eukaryotes, these HEPN proteins process RNAs on the inner side of the membrane. The specialized TM segments with the WP signature and the potential external domains could act as sensors for stimuli on the cell surface, and the resulting signal could affect the HEPN domain conformation and hence RNA stability. We also identified HEPN domains that are fused to CBS domains, in some cases together with additional HD phosphohydrolase domains [Pelobacter propionicus; Figure In the present work, we identified at least three distinct groups of HEPN domains that are combined with TM segments. The first of these belongs to the family that overlaps with the Pfam DUF4145 family  fused to DEATH domains, the key adaptors in metazoan apoptosis and immunity [Bacillus cereus) that often combine an N-terminal HEPN domain with NTPase modules of the STAND superfamily , and inT NTPase .Plasmodium falciparum was found to be conserved throughout apicomplexa. These proteins combine a pair of N-terminal aegerolysin domains with C-terminal HEPN domains  and suicide/dormancy-based defenses . The repThe present analysis of the HEPN domain also supports the recent hypothesis that prokaryotic intra- and inter-genomic conflict systems provided raw material for the emergence of new core cellular functions in eukaryotes . The emeThe findings presented here are expected to instigate and guide laboratory experiments that have the potential to illuminate numerous aspects of cellular biochemistry and biology across the three domains of life.http://ftp.ncbi.nih.gov/blast/documents/blastclust.html) to cluster sequences at different thresholds. Multiple sequence alignments were built using the Kalign [http://ftp.ncbi.nih.gov/blast/documents/blastclust.html) to identify related sequences in gene neighborhoods. Each cluster of homologous proteins were then assigned an annotation based on the domain architecture or conserved shared domain. This allowed an initial annotation of gene neighborhoods and their grouping based on conservation of neighborhood associations. The remaining gene neighborhoods were examined for specific template patterns such as TA systems. In this analysis care was taken to ensure that genes are unidirectional on the same strand of DNA and shared a putative common promoter to be counted as a single operon. If they were head to head on opposite strands they were examined for potential bidirectional promoter sharing patterns. We also filtered the data using an intergenic distance criterion of 100 nt for genes to belong to a predicted operon. A complete list of Genbank gene identifiers for proteins investigated in this study is provided in the Additional file Iterative profile searches with the PSI-BLAST  and JACKe Kalign , MUSCLE e Kalign  and PCMAe Kalign  programse Kalign . Remote e Kalign . Gene neThis is a strong, encyclopedic survey and analysis of a large and diverse family of important protein domain families. The search strategy was quite clever. Dealing with remote homologs is never easy and the authors did an excellent job in finding them and then proving their relatedness using extensive profile-profile comparisons and structural considerations. The results lay foundation for future experimental studies in this area, especially when existing domain models in public databases will be appropriately changed. I have decided not to list minor technical points, especially because 50 pages without line and page numbering are hard on a reviewer, and I have only a couple of suggestions to offer:1. The title sounds as the authors have just discovered the HEPN domain, which is obviously not the case. There should be something in the title  that correctly states their findings. We have changed the title as suggested.Response:2. Better placement of the new results into the Pfam context would be helpful. HEPN is currently listed as a domain (PF05168) within the nucleotidyltransferase substrate- binding domain clan , not HEPN clan, as stated by authors. Yes this is indeed the case. We have now corrected the text to reflect the Pfam nomenclature accurately.Response:Authors indicated that their searches retrieved proteins that belong to three families of this clan in addition to HEPN: DUF4145, DUF86 and “C-terminal domains of several polymerase β–superfamily proteins”. The Pfam clan also includes GlnD_UR_UTase, NTase_sub_bind, PaREP1, and DUF294_C domain families. Their relationship to authors’ results remains unclear: are they all “C-terminal domains of several polymerase β–superfamily proteins”? Regardless of the answer, this relationship should be better explained. The Pfam models labeled GlnD_UR_UTase, NTase_sub_bind and DUF294_C are C-terminal domains of the polymerase β–superfamily. We have now modified the text to state clear which Pfam models correspond to the several polymerase β–superfamily proteins we mention in the text. In Table 1 we provide a complete mapping of the families to Pfam models. This provides the relationship to our results in terms of new families which we identified.Response:The authors present a very comprehensive analysis of the HPEN domain. I appreciate the range of the conclusions that the authors arrive at, from molecular function to evolutionary patterns. The multiplicity of observations and predictions make it very hard to come up with any constructive criticism. Most of the predictions are short stories that may well be true, but about which I do not have enough knowledge to comment upon. So I will only make some short, specific remarks.Methodological: I cannot really find how the authors decided whether genes are in the same operon, e.g. for the operons depicted in Figure  We have now added details regarding how this was done in the Methods section. Briefly, we used two criteria: 1) proximity and 2) conservation of gene-neighborhoods across phylogenetically distant groups of prokaryotes. This data was extracted for each genome using the RefSeq database when possible or the whole-genome-shotgun sequence when the respective genome was missing in RefSeq. We used the gene annotations supplied by the sequencing centers to determine the intergenic distances.Response:References: References 64 and 65 are not original references to genes occurring with conserved gene order, or in operons, having related functions. We have now added appropriate references to the original works that describe the method of conserved gene-neighborhoods in contextual inference.Response:Figures: showing both Figure  We still believe that displaying both depictions has some value because the first reflect the “natural” clustering based on the Kamada-Kawai algorithm, whereas the second is the knowledge-based manual clustering of functions. Showing both helps highlight the fact that there is considerable contextual information that allows discernment of these functional categories from the data itself.Response:If the authors want the reader to see the circular permutation in the KEN domain they may have to show it better. To me it is not obvious. We have now modified the figure with the N- and C- terminus labeled in order to depict the permutation clearly.Response:“The conserved acidic residue” (page 9) is, I take it, part of the second motif mentioned earlier. Please repeat the name of that motif so that the reader knows what is meant” We have modified the sentence to clarify the sameResponse:All other editorial changes which were suggested were made.Did not provide any comments.The authors declare that they have no competing interests.KM, VA, AMB, and LA collected data; KM, VA, EVK and LA analyzed the data; LA and EVK wrote the manuscript that was read and approved by all authors.Provides access to: 1) comprehensive list of Genbank identifiers, architectures and operons and table of HHpred search probability percentages. 2) A comprehensive raw alignment of HEPN domains. 3) NTD domains combined with HEPN and other defense related domains. 4) Associated domains described for the first time in this work.Click here for file


Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate  S. cerevisiae. Metazoans have large numbers of SH2, PTB, tyrosine kinase, and tyrosine phosphatase domains, suggesting a concerted expansion of the apparatus needed for phosphotyrosine signaling. Surprisingly, the genome of the unicellular choanoflagellate Monosiga brevicollis contains numbers of each of these domains that are comparable to complex multicellular organisms Monosiga brevicollis genome affords an important glimpse into the early evolution of tyrosine kinases and phosphatases.The machinery necessary for phosphotyrosine-based signaling in metazoans includes “writer” domains (tyrosine kinases), “readers” (SH2 and PTB domains), and “erasers” (tyrosine phosphatases) Monosiga brevicollis display combinations of domains that are not observed in multicellular animals Monosiga brevicollis (MbSrc1 and MbSrc4) have suggested that the substrate targeting function of the SH3 and SH2 domains evolved earlier than the ability of the domains to engage in autoinhibitory interactions In addition to their catalytic domains, metazoan nonreceptor tyrosine kinases (NRTKs) possess noncatalytic domains that are important in kinase function Monosiga brevicollis genome contains fifteen HMTK kinases HM motif-containing tyrosine kinase, so named because the conserved His-Arg-Asp sequence within the catalytic loop is replaced by a His-Met sequence in this family). Ten of the fifteen HMTK kinases contain one or more PTB domains, which in multicellular organisms often bind to phosphotyrosine-containing proteins The 6 was from Covance, and anti-GST was from Molecular Probes. Horseradish peroxidase-linked sheep anti-mouse IgG antibodies were from GE Healthcare. Leupeptin, aprotinin, PMSF, sodium vanadate, sodium fluoride, pyruvate kinase/lactate dehydrogenase enzymes, reduced NADH, ethanolamine, and EZview red anti-Flag M2 affinity gel were from Sigma. Affi-gel 15 agarose was purchased from BioRad.Anti-phosphotyrosine antibody (clone 4G10) was from Millipore, anti-Flag M2 and anti-tubulin clone GTU-88 were from Sigma, mouse monoclonal anti-HisM. brevicollis cDNA library ATCATGGGCGTCTTTGAAGCCACC and the 3′ primer GCTCTAGATCAATTCCTGTGCC-ATGTTGGCAAAGGATGGGCG. The 5′ primer binds at the start of the third PTB domain of HMTK1 (residue 341); we were not successful in amplifying a cDNA using primers at the 5′ end of the gene model, or at the beginning of the first or second PTB domains. The 3′ primer extends to Leu 761 of the predicted sequence. PCR reactions using a 3′ primer based on the entire 784 amino acids were unsuccessful. Thus, the 23 C-terminal amino acids from the gene model are missing. These residues are not predicted to be in a conserved domain.The predicted sequence of HMTK1 (784 amino acids) was obtained from the Joint Genome Institute gene model (NCBI accession number: XM_001749555). The form of HMTK1 used in this study was amplified by PCR from a EcoRI site of pFastbac-Hta (Invitrogen). FLAG-tagged HMTK1 was expressed in mammalian cells by subcloning into the BglII and BamHI sites of p3XFLAG-CMV (Sigma). The ΔPTB version of this construct was prepared by PCR amplification and subsequent recloning into the BamHI and EcoRI sites of p3XFLAG-CMV. Site-directed mutagenesis of FLAG-HMTK1 was carried out using the QuikChange kit (Stratagene). To express the isolated third PTB domain (residues 341–474) in bacterial cells, this PCR fragment was cloned into the EcoRI site of plasmid pGEX-4T-1 . All constructs were confirmed by DNA sequencing.For expression in insect cells, HMTK1 DNA (encoding residues 341–761) was cloned into the Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac system (Invitrogen). Sf9 cells (800 ml) were infected with HMTK1 baculovirus and harvested after three days. Cells were lysed in a French pressure cell, and His-tagged HMTK1 was purified using nickel-nitrilotriacetic acid resin (Qiagen), as described previously E. coli cells (1 liter) expressing the GST fusion protein were lysed in a French pressure cell, and the GST-PTB protein was purified by chromatography on a glutathione-agarose column (Molecular Probes).HMTK1 was expressed in 2, 0.2 mM ATP, [γ-32P]-ATP (30–50 cpm/pmol), and varying amounts of HMTK1 and peptide substrates. All peptide substrates were purified by high pressure liquid chromatography and characterized by mass spectrometry prior to use. The following substrates were used: c-Src peptide, AEEEIYGEFEAKKKG; MbSTAT peptide, KKKASGYVMADIA; RTKB2 peptide 1, SEEVYGAVVDKKK; RTKB2 peptide 2, AEEVYEAIADKKK; SH2 binding substrates (varying linker lengths), RRLEDAIYAAGGGGGEPPQpYEEIG, RRLEDAIYAAGEPPQpYEEIG, and RRLEDAIYAPQpYEEIG; SH2 control substrate, RRLEDAIYAAGGGGGEPPQFEEIG. Initial rate kinetic measurements on the PTB ligand-containing substrate (KKAEEEIYGEFEANFTNPVpYATLG) and on the Phe-containing PTB control (KKAEEEIYGEFEANFTNPVFATLG) were carried out using a continuous spectrophotometric assay 2, 2 mM ATP, 1.5 mM phosphoenolpyruvate, 90 units/ml of pyruvate kinase, 109 units/ml of lactate dehydrogenase, and 1.2 mg/ml of NADH. For determination of Km values, the enzyme concentrations were 0.3–0.5 µM and the peptide concentrations ranged from 0–700 µM. Kinetic constants were determined by nonlinear fitting to the [substrate] vs. velocity curves using GraphPad Prism 4.Kinase assays were performed by two methods. Initial activity measurements were performed by the phosphocellulose paper binding assay 3VO4). Bound proteins were eluted with SDS-PAGE loading buffer, separated by 10% SDS-PAGE, and visualized either by Coomassie blue staining or by Western blotting.The PTB ligand-containing peptide, Phe-containing PTB control, and pYEEI peptide (EPQpYEEIG) were linked to Affi-Gel 15 resin (Bio-Rad) following the manufacturer's protocols. Purified HMTK1 or the isolated PTB domain were incubated for 1 hour at 4°C with the immobilized peptides. After binding, the beads were washed 4 times with 1 ml of binding buffer  that is part of the linker region between the GST and PTB domains. The resulting pGEX-BiotinN-PTB construct was transformed into E. coli BL21(DE3) cells for expression. GST-biotin-PTB protein was purified using glutathione-agarose and desalted using a PD-10  gel filtration column.The PTB domain of HMTK1 was cloned as an EcoR1 fragment into a modified pGEX  bicistronic vector pGEX-4T-BiotinN to express the gene of interest and the biotin ligase (BirA) in the same cell  on cellulose based matrix (Jerini Biotools). To ensure protein accessibility, each peptide contained a 4-residue spacer consisting of a glycine and 3 β-alanines. All peptides were immobilized via their C-termini. To assess synthesis reproducibility, six different phosphopeptide sequences predicted for 14-3-3 binding were made in duplicate in different rows on the array, and displayed less than 10% standard error for 14-3-3 binding levels between identical sequences. Prior to usage, peptide array membranes were washed in binding buffer  and blocked with 5% filtered bovine serum albumin in the same buffer. Recombinant biotinylated PTB protein was diluted to 10 µg/ml in binding buffer containing 5% BSA, and incubated with the array membrane for 30 min at room temperature. After washing (2×10 min), streptavidin-HRP  was added for 15 minutes at RT. After washing in binding buffer (3×10 min), bound PTB was detected using enhanced chemiluminescence and standard X-ray film.6) were plated in 15-cm diameter dishes and transfected after 24 hours using 5–15 µg of DNA and 2 µl TransIT per µg DNA. After 48 hours, the cells were harvested, washed twice with PBS and lysed using radioimmune precipitation assay (RIPA) buffer  supplemented with the protease inhibitors leupeptin (10 µg/ml), aprotinin (10 µg/ml), PMSF (200 µM) and the phosphatase inhibitors Na3VO4 (0.2 mM) and NaF (10 mM). Lysates (50 µg) were separated by 10% SDS-PAGE, transferred to PVDF membranes and probed with the appropriate antibodies. For immunoprecipitation, cell lysates (100 µg) were incubated overnight at 4°C with 40 µl of EZview red anti-Flag M2 affinity gel pre-equilibrated with RIPA buffer in a total volume of 1 ml. The immunocomplexes were washed 4 times, eluted from the beads by adding Laemmli buffer, separated by SDS-PAGE, transferred to PVDF membranes and probed with the appropriate antibodies.SYF cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Sigma) and 1000 UI/ml penicillin, 1000 UI/ml streptomycin and 25 ng/ml amphotericin B . SYF cells , with the N-terminal residues to the tyrosine typical for autophosphorylated acidophilic kinases. However, the conserved HRD motif in the catalytic loop is replaced with HMD . The kinase domain is most closely related to the fibroblast growth factor receptor-1 tyrosine kinase  cells. We purified the His-tagged PTB-kinase construct using nickel-nitrilotriacetic acid resin. For our initial enzymatic characterization, we measured phosphorylation of an acidophilic Src peptide (AEEEIYGEFEAKKKKG) 32P-labeled ATP (M. brevicollis kinase substrates. Two of the peptides (RTKB2 peptides 1 and 2) correspond to sequences from the intracellular domain of a M. brevicollis receptor tyrosine kinase, and the third (MbSTAT) is from a putative M. brevicollis STAT Monosiga kinases previously characterized (MbSrc1 and MbSrc4) had substantially higher activities towards RTKB2-1 and RTKB2-2 compared with the c-Src peptide To test whether HMTK1 is enzymatically active, we cloned the HMTK1 DNA into a baculovirus expression vector and expressed the enzyme in eled ATP . HMTK1 p2 under conditions that typically promote autophosphorylation of tyrosine kinases  is comparable to the value for Src family kinases with the same peptide , showed binding to the biotinylated PTB domain that was strongly phosphotyrosine-dependent . We syntMonosiga brevicollis likewise shows numerous examples of tyrosine kinase catalytic domains connected to modular signaling domains Monosiga have not been seen elsewhere, including in metazoans. The noncatalytic regions of these kinases presumably have important functional roles, similar to their metazoan counterparts.Metazoan nonreceptor tyrosine kinases invariably possess noncatalytic modular domains in addition to their conserved catalytic domains. These noncatalytic regions play important roles in subcellular localization, enzymatic regulation, protein-protein interactions, and substrate recognition Monosiga HMTK kinases contain at least one PTB domain, although some of the domains are small and may not fold into a functional unit. Although the number of PTB domains varies in this family (1–3 domains), it is noteworthy that the PTB domains all lie N-terminal to the kinase domains, i.e., in the same relative position as SH2 domains and kinase domains in Src-family kinases. In Src, the positioning of the SH2 domain relative to the kinase domain plays a role in substrate recognition in vitroHere, we have carried out a biochemical study of one such kinase, HMTK1. Ten of the fifteen Monosiga HMTK kinases  show the most similarity to these same families of PTB domains, suggesting that HMTK1 is fairly typical of the family in this regard. The HMTK1 catalytic domain is most closely related to mammalian receptor tyrosine kinase domains.The combination of a PTB and a tyrosine kinase domain is not found in higher metazoans. On the other hand, the individual PTB and kinase domains of HMTK1 show sequence conservation with related domains from metazoans. The third PTB domain of HMTK1  are related to the Numb and Gulp families of PTB domains . PTB domMonosiga. The activity of HMTK1 was undetectable in these cells . Our data cannot distinguish between these possibilities at present.We confirmed that HMTK1 possesses tyrosine kinase activity by expressing the protein in insect cells, purifying it, and measuring phosphorylation of synthetic peptides. To examine HMTK1 activity in intact cells, we turned to a heterologous cell system , due to the difficulties associated with expressing genes in se cells . We alsom, which is comparable to the effects of the pYEEI motif recognized by the Src SH2 domain.We carried out experiments with immobilized peptide arrays to search more broadly for HMTK1 binding partners. Pawson and coworkers previously used NPXY peptide arrays to screen for binding partners for 10 diverse PTB domains Monosiga brevicollis cells. To identify potential substrates, we searched for occurrences of the TNFTNPVYATLG motif within the Monosiga genome using the protein-translated nucleotide BLAST search tool on the genome site (http://genome.jgi-psf.org/Monbr1/Monbr1.home.html). Although there were no matches to the full sequence, two predicted proteins had partial matches. Gene model number 11339 encodes a predicted transmembrane protein with cadherin and SH2 domains; the predicted protein includes a FSNPMYA sequence. A second SH2-containing Monosiga protein (gene model number 34447) contains the sequence NPVYA. We carried out a similar analysis using other PTB-binding peptides from the array experiment. The sequence EYGEL from Monosiga RTKB8 was identified by a search using peptides 11/12 . MAP kinases, for example, possess distal docking sites that enhance substrate specificity Figure S1Sequence alignment of the kinase domains of HMTK1 and human fibroblast growth factor receptor 1. The HRD/HMD sequences in the catalytic loop are underlined, and the tyrosine residue in the predicted activation loop is shown in red.(EPS)Click here for additional data file.Figure S2Autophosphorylation of HMTK1. Purified HMTK1 (36 ng) was analyzed by anti-pTyr Western blotting after incubation with Yersinia tyrosine phosphatase (+YOP), with no treatment (HMTK), or after an autophosphorylation reaction (+ATP). The autophosphorylation reaction contained 1 mM ATP and 10 mM MgCl2, and proceeded for 45 minutes at 30°C.(EPS)Click here for additional data file.Figure S3Structural model of HMTK1 PTB. A homology model of the third PTB domain of HMTK is shown, based on the human Numb-R protein http://toolkit.tuebingen.mpg.de/hhpred). Numb-R contains two Phe residues that are important for ligand binding (EPS)Click here for additional data file.Figure S4Expression of HMTK1 in mammalian cells. (A). SYF cells were transfected with FLAG-tagged HMTK1 . Whole cell lysates were analyzed by SDS-PAGE and Western blotting with anti-pTyr, anti-FLAG, or anti-tubulin antibodies, as indicated. C = untransfected SYF cells. The figure is representative of 4 experiments. (B) Similar experiments were carried out on control or HMTK1-transfected SYF cells treated with 0. 0.1, or 1.0 mM sodium orthovanadate  prior to lysis. Similar amounts of lysates were used in panels (A) and (B), but a shorter exposure time was used in (B) than in panel (A).(EPS)Click here for additional data file.Figure S5Immunoprecipitation experiment. HMTK1 (wild-type or mutants) was immunoprecipitated from SYF cells using anti-FLAG affinity resin. Proteins in the immunocomplexes were separated by SDS-PAGE and analyzed by Western blotting with anti-pTyr and anti-FLAG antibodies. The band at ≈110 kDa is a non-specific band. The figure is representative of 3 experiments.(EPS)Click here for additional data file.


Proteins are composed of domains, protein segments that fold independently from the rest of the protein and have a specific function. During evolution the arrangement of domains can change: domains are gained, lost or their order is rearranged. To facilitate the analysis of these changes we propose the use of multiple domain alignments.We developed an alignment program, called MDAT, which aligns multiple domain arrangements. MDAT extends earlier programs which perform pairwise alignments of domain arrangements. MDAT uses a domain similarity matrix to score domain pairs and aligns the domain arrangements using a consistency supported progressive alignment method.MDAT will be useful for analysing changes in domain arrangements within and between protein families and will thus provide valuable insights into the evolution of proteins and their domains. MDAT is coded in C++, and the source code is freely available for download at http://www.bornberglab.org/pages/mdat.The online version of this article (doi:10.1186/s12859-014-0442-7) contains supplementary material, which is available to authorized users. Proteins are composed of domains, i.e. amino acid segments which have a specific function and/or a structure, fold independently from the rest of the protein and are evolutionary well conserved -3. DomaiPlasmodium falciparum are probably the result of a trade-off between optimizing within-host fitness and minimizing between-host immune selection pressure [Several studies have shown the importance of changes in domain arrangements during evolution. New arrangements can be produced by shuffling of existing domains. These new arrangements played, for example, an important role during the evolution of vertebrates where they are involved in vertebrate specific structures like the cartilage . In addipressure . Also, tpressure .The best currently available methods to study domain arrangements are classical multiple sequence alignment (MSA) methods, for example T-Coffee  or ClustThere are several advantages in using MDAs instead of MSAs. Due to the much shorter arrangement length compared to the primary sequence, an MDA can be calculated faster and with lower memory requirements, which is especially an advantage with large datasets. Another advantage is that a domain arrangement is more conserved than the underlying amino acid sequence. It is therefore possible to produce meaningful MDAs when the amino acid sequences are already too divergent to be compared. Furthermore, it is easier to visually examine the resulting alignments, due to the smaller number of characters.Since domain arrangement similarity and differences can provide insights into functional similarity and changes between proteins (see above) we present an algorithm which helps to compute an MDA and facilitate the analysis of domain arrangements of different proteins. In this paper, we present MDAT (Multiple Domain Alignment Tool), a program that takes multiple domain arrangements and aligns them. It uses a domain similarity matrix reflecting the similarity between all pairs of domains in the Pfam database. Using a combination of the RADS  algorithThe Pfam database provides some rough information on domain homology (the “clans”) based on a range of various information evaluated manually ,20. UnfoAs expected, most domain pairs have a low probability of being a true positive match. It is interesting to note that a high number of domain pairs coming from the same clan have a very low probability of being a true match and that at least some domain pairs from different clans or without clan assignment have a high probability of being a true match.We use the BAliBASE3  benchmarFer2 domains together, while MAFFT only aligned 4 of them. Moreover, these domains are not kept as unit but split up into parts that are stretched along the whole alignment. Another example can be viewed in Additional file An example for the advantage of using domains in the construction of alignments can be seen in Figure The use of domains as anchor-points can strongly reduce the memory usage and running time. Table We show that using MDAs themselves has its merits. MDAs can be used to visualize in a simple way the similarity between domain arrangements. Just like any alignment program, MDAT is not able to handle inversions. However, due to the low number of domains in a protein, inversions can be easily detected in a graphical view, which in not possible at the amino acid level. Furthermore, we demonstrate that an MDA is a good starting point for a multiple sequence alignment. It is particularly useful as guidance for the MSA, because it strongly increases the speed with which a multiple sequence alignment is calculated. Currently, the resulting MSAs from MDAT are not as accurate as traditional sequence alignments, however, due to the short calculation time, we are able to handle larger data sets. For many analyses, such as genome projects, the detection of domains is an essential part of the standard annotation procedure. Therefore, domain annotation is often readily available.2 alignment pairs. As recommended [Contrary to amino acids, no scoring matrix currently exists to handle domain matches. Therefore, we calculated a domain similarity matrix (DSM) for Pfam-A domains that stores a similarity value for each domain pair. The entries of the DSM are calculated using the HHsearch  program.ommended , we usedommended  and PAM ommended  matricesDomain collapsing: Given a set of domain arrangements, the first step of the MDA construction is to collapse identical domain arrangements into a single one. A set of identical domain arrangements is from here on represented with a single arrangement. The length of a domain in this representative arrangement is defined as the average of the domains it contains. A change in tandem domain repeats is the most frequently occurring domain rearrangement event . Tandem Library construction: In the next step the RADS algorithm, a dynamic programming algorithm, is used to produce alignments between all pairs of arrangements. RADS has been extended to use the DSM to score a match of two domains instead of a fixed value. The matches identified in this alignment are then stored in a library.α in arrangement X and domain β in arrangement Y are matching, then the score of the match α−β is increased if there are arrangements Z with domain γ that is matching α as well as β.Library extension: The matches from the library are rescored according to the algorithm described in T-Coffee. The reweighting has the purpose to increase the score of a match that is supported by matches in a third sequence: If domain Alignment calculation: Using these scores a normal progressive alignment, as first described by Higgins and Sharp , is perfRefinement: The last step of the algorithm is a simple refinement step. Blocks of domains are shifted to other columns if this increases the number of identical domains in a column.The MDA is constructed in several steps and is based on the RADS  pairwiseAn MDA can be used to guide the MSA alignment process. MDAT uses a similar approach to the one described in Dialign-Pfam. Blocks of domains are used as anchor points to limit the search space and thereby increasing the speed of the alignment calculation. Furthermore, using the MDA as an anchor, guarantees that the correct domains are aligned with each other see . Unlike First alignment step: All sequences that are represented by the same domain arrangement are aligned first. The sequences are split at domain boundaries and each pair of segments is aligned separately, thus allowing easy parallelization of this step. Domain segments are aligned using a banded alignment approach as previously described in the Pecan genome aligner .Second alignment step: Following a guide tree constructed from the domain architecture similarity, the alignment profiles computed in the first step are progressively aligned with each other. At each node in the tree, two profiles are aligned that are based on different domain architectures. Similar to the first step, sequences can be split at domain boundaries. However, this is only possible at domains that are aligned with each other. The sequence segments between two aligned domains cannot be simply aligned in a global fashion, because there may be non-aligned domains which should not be aligned on the sequence level either   and Clus