鼻咽癌组织miR-20b-5p、miR-325-3p表达水平与放疗敏感性和预后的关系研究

摘要 目的：探讨鼻咽癌组织微小核糖核酸（miR）-20b-5p、miR-325-3p表达水平与放射治疗敏感性和预后的关系。方法：选取2017年11月至2019年6月我院收治的84例确诊为鼻咽癌并拟进行放射治疗的患者设为鼻咽癌组,另选取同期收治的42例慢性鼻咽炎患者为对照组,比较鼻咽癌组织及鼻咽部炎症组织中miR-20b-5p、miR-325-3p表达水平,分析鼻咽癌组织中miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者临床病理特征的关系。根据鼻咽癌患者放疗敏感性评估结果分为敏感组和抵抗组,比较两组miR-20b-5p、miR-325-3p表达水平。随访3年,Kaplan-Meier法及Cox回归分析法分析miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者生存预后的关系。结果：鼻咽癌组miR-20b-5p、miR-325-3p表达水平均高于对照组（P＜0.05）。不同T分期、N分期、临床分期患者在miR-20b-5p、miR-325-3p高表达组与低表达组中的占比比较存在统计学差异（P＜0.05）。完成7~8周放疗后3个月评估患者放疗抵抗率36.90%,抵抗组miR-20b-5p、miR-325-3p表达水平均高于敏感组（P＜0.05）。miR-20b-5p高表达鼻咽癌患者的累积生存时间短于miR-20b-5p低表达患者（P＜0.05）;miR-325-3p高表达鼻咽癌患者的累积生存时间短于miR-325-3p低表达患者（P＜0.05）。单因素、多因素Cox回归分析显示,年龄＞60岁、T3/T4期、miR-20b-5p高表达、miR-325-3p高表达是鼻咽癌患者预后不良的独立危险因素（P＜0.05）。结论：鼻咽癌组织中miR-20b-5p、miR-325-3p均异常高表达,其表达水平与肿瘤浸润深度、淋巴结转移、临床分期及放疗敏感性有关,且miR-20b-5p、miR-325-3p高表达患者放疗后预后不良风险更大。     

Cytologically balanced t(2;20) in a two-generation family with alagille syndrome: cytogenetic and molecular studies.

Alagille syndrome is a clinically defined, dominantly inherited disorder affecting the liver, heart, face, eye, and vertebrae. Alagille syndrome has previously been localized to the short arm of chromosome 20, on the basis of reports of a small number of patients with chromosomal deletions of 20p. We undertook a cytogenetic study of patients with Alagille syndrome and identified a family in which a cytologically balanced translocation between chromosomes 2 and 20, 46,XX/XY, t(2;20)(q21.3;p12), is segregating concordantly with the disease. The breakpoint on chromosome 20p in this t(2;20) is consistent with the shortest region of overlap demonstrated in the reported deletion patients. This is the first report of a translocation associated with 20p and Alagille syndrome, and this rearrangement confirms the location of the Alagille disease gene at 20p12. We have established a somatic cell hybrid from a lymphoblastoid cell line from one of the affected individuals that contains the derivative chromosome 20 (20qter-->p12::2q21.3-->qter) but not the derivative chromosome 2, the normal chromosome 2, or the normal chromosome 20. Southern blot and PCR analysis of probes and sequences from 20p have been studied to define the location of the translocation breakpoint. Our results show that the breakpoint lies distal to D20S61 and D20S56 within band 20p12.     

Sex and stride length impact leg stiffness and ground reaction forces when running with body borne load

This study quantified leg stiffness and vGRF measures for males and females using different stride lengths to run with four body borne loads (20, 25, 30, and 35 kg). Thirty-six participants (20 males and 16 females) ran at 4.0 m/s using either: their preferred stride length (PSL), or strides 15% longer (LSL) and shorter (SSL) than PSL. Leg stiffness and vGRF measures, including peak vGRF, impact peak and loading rate, were submitted to a RM ANOVA to test the main effect and interactions of load, stride length, and sex. Leg stiffness was greater with the 30 kg (p = 0.016) and 35 kg (p < 0.001) compared to the 20 kg load, but decreased as stride lengthened from SSL to PSL (p < 0.001) and PSL to LSL (p < 0.001). Males exhibited greater leg stiffness than females with SSL (p = 0.029). Yet, males decreased leg stiffness with each increase in stride length (p < 0.001; p < 0.001), while females only decreased leg stiffness between PSL and LSL (p = 0.014). Peak vGRF was greater with the addition of body borne load (p < 0.001) and increase in stride length (p < 0.001). Both impact peak and loading rate were greater with the 30 kg (p = 0.034; p = 0.043) and 35 kg (p = 0.004; p = 0.015) compared to the 20 kg load, and increased as stride lengthened from SSL to PSL (p = 0.001; p = 0.004) and PSL to LSL (p < 0.001; p < 0.001). Running with body borne load may elevate injury risk by increasing leg stiffness and vGRFs. Injury risk may further increase when using longer strides to run with body borne load.     

Molecular and cytogenetic analysis of an interstitial 20p deletion associated with syndromic intrahepatic ductular hypoplasia (Alagille syndrome)

Summary High-resolution chromosome analysis of a 19-year-old female proband with syndromic intrahepatic ductular hypoplasia (Alagille syndrome, AWS) revealed an interstitial deletion of chromosome 20p with breakpoints provisionally located in or close to p11.22 and p12.2. Southern blots from digests of DNA of the proband and her chromosomally normal parents were hybridized with the human DNA probes pR12.21, HuPrPcDNA2, and pDS6-SgI, which have been mapped to the region 20 (p12-pter), and rehybridized with the F IX probe for calibration. Comparing the hybridization signals of the normally sized DNA fragments of the familiy, we found no evidence for loss of any of the three tested distal chromosome 20p loci in our proband. Furthermore, in situ hybridization with HuPrPcDNA2 revealed a specific accumulation of grains at or around the faint distal G band suspected to represent all or most of band p12.3 of the proband's deleted 20p and at p12 of the normal chromosome 20. Thus the AWS of our proband is associated with an interstitial deletion that preserved the three tested distal loci on 20p. Since nine further reported cases of 20p deletion are clinically similar, we propose AWS as a further contiguous gene syndrome and assign it to an approximately 8-Mb-large chromosome 20p segment (provisionally, p11.23–p12.1).     

Impacts of CLA and dietary concentrate proportion on blood metabolite concentration and proliferation of peripheral blood mononuclear cells of periparturient dairy cows

The study aimed to examine effects of supplemented CLA to periparturient dairy cows receiving different concentrate proportions antepartum (a.p.) to investigate CLA effects on metabolism and immune function. Compared with adapted feeding, high-concentrate diet a.p. should induce a ketogenic metabolic situation postpartum (p.p.) to better understand how CLA works. A total of 64 pregnant German Holstein cows had ad libitum access to partial mixed rations based on concentrate and roughage 3 weeks before calving until day 60 p.p. A.p., cows received 100 g/day control fat (CON) or a CLA supplement, either in a low-concentrate (20%, CON-20, CLA-20) or high-concentrate diet (60%, CON-60, CLA-60). P.p., concentrate proportion was adjusted to 50% while fat supplementation continued. After day 32 p.p., half of the animals of CLA-groups changed to CON supplementation (CLA-20-CON, CLA-60-CON). A ketogenic metabolic state p.p. was not achieved and respective impacts of CLA could not be examined. Blood samples for isolation of peripheral blood mononuclear cells (PBMC) were collected on day −21, 7, 28 and 56 relative to calving. Blood chemistry samples were taken over the entire experimental period. Mitogen-stimulated proliferation of PBMC remained unaffected. Besides serum concentrations of triglycerides, total bilirubin, total protein, albumin and IGF-1, clinical-chemical serum characteristics remained uninfluenced by treatments. No post-supplementation effect could be observed. Measured blood metabolites and mitogen-stimulated proliferation of PBMC indicate that all groups had an increased metabolic stress around calving, whereby group CLA-20 was affected more severely. Overall, supplemented CLA did not positively affect metabolism or immune function of periparturient dairy cows. However, feeding CLA in a low-concentrate diet a.p. seems to increase liver stress around calving via reduced DMI.     

Ascocarp production by Nannizzia otae on keratinous and non-keratinous agar media and mating behavior of N. otae and 123 Japanese isolates of Microsporum canis

Ascocarp production byNannizzia otae, VUT 77054+x VUT 77055–, was compared on 8 different (1 keratinous and 7 non-keratinous) agar media.Oatmeal salts agar and diluted Sabouraud dextrose salts agar with or without yeast extract were found to be unsuitable for ascocarp production in this species. In contrast, three different variants of oatmeal salts agar enriched with yeast extract proved to be satisfactory for the same purpose, while oatmeal salts agar with both yeast extract and horsehair powder was not necessarily superior to the former three media in this regard. Niger seed salts agar enriched with yeast extract was the most superior to any other seven media in all of the following respects; i.e., the number of gymnothecia produced per plate, the germination rate of ascospores, and the suppression of sporulation.Asci from the cross VUT 77054+ x VUT 77055– that yielded abundant fertile gymnothecia on niger seed salts agar with yeast extract were dissected and 51 ascospores were randomly isolated. Of the 51 ascospores, 47 (92%) germinated to form mature colonies. Of the 47 monoascospore F₁ progeny back crossed to the parentals, 21 (45%) mated or reacted with the + mating type, 18 (38%) did with the – mating type, and the remaining 8(17%) were sexually nonreactive.One hundred and twenty-three Japanese isolates ofMicrosporum canis, obtained from human and animal ringworms for the past 12 years, were also crossed with the tester strains ofN. otae on selected 3 of the 8 media to determine their mating type. Out of these 123, 113 (92%) produced fertile gymnothecia in crosses with VUT 77054 +, 9(7%) were non-reactive, and the only one isolated from human in Osaka city produced fertile gymnothecia in crosses with VUT 77055–. The data suggest thatM. canis (N. otae) exists predominantly as – mating type in Japan. A possible explanation for this unequal distribution of mating type is presented.     

A novel family-specific translocation t(2;20)(p24.1;q13.1) associated with recurrent abortions: molecular characterization and segregation analysis in male meiosis

In the present study, we present a novel reciprocal translocation t(2;20)(p24.1;q13.1) and its segregation in a three generation family. The rate of miscarriages (50%) in pregnancies from male translocation carriers could be explained by unbalanced translocation-bearing spermatozoa found with a frequency of approximately 55% in the entire sperm population of a t(2;20)(p24.1;q13.1) carrier. These imbalanced spermatozoa mainly present as 2, der(20) and der(2), 20 missegregated (approximately 46%) while adjacent 2 and 3:1 segregation patterns account for approximately 5% and 4% of imbalances, respectively. While the translocation is associated clearly with an increased risk of early abortions (7/12) in both male and female carriers, no malformed livebirths were observed. Our results suggest complete embryonic lethality of imbalanced offspring. With respect to a high rate of segregation to 2, der(20) and to der(2), 20 imbalanced spermatozoa in male translocation carriers and with respect to known cases of partial trisomy 2p and 20q we consider that their corresponding monosomies result in fetal loss. This is the first study reporting multiple abortions associated with partial monosomy 20q13.1-->qter and 2pter-->p24.1 and the first report on the frequency of chromosomal imbalances in gametes of a male t(2;20)(p24.1;q13.1) heterozygote.     

Licochalcone A reverses NNK-induced ectopic miRNA expression to elicit in vitro and in vivo chemopreventive effects

BackgroundChemoprevention is the best cost-effective way regarding cancers. MicroRNAs (miRNAs) have been reported to be differentially expressed during the development of lung cancer. However, if lung cancer prevention can be achieved through modulating miRNAs expression so far remains unknown.PurposeTo discover ectopically expressed miRNAs in NNK-induced lung cancer and clarify whether Licochalcone A (lico A) can prevent NNK-induced lung cancer by modulating miRNA expression.Study design and methodsA/J mice were used to construct a lung cancer model by intraperitoneal injection with physiological saline NNK (100 mg/kg). Chemopreventive effects of lico A against lung cancer at 2 mg/kg and 20 mg/kg doses were evaluated in vivo. MicroRNA array and RT-qPCR were used to assess the expression levels of miRNAs. MLE-12 cells were treated with 0.1 mg/ml NNK, stimulating the ectopic expression pattern of miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p. miR-144-3p mimics and inhibitors were used to manipulate miR-144-3p levels. The effects of lico A (10 μM) on cell cycle distribution, apoptosis, and the expression of CK19, RASA1, miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p in NNK-treated MLE-12 cells were studied.ResultsThe expression levels of miR-144-3p, miR-20a-5p, and miR-29c-3p increased, while those of let-7d-3p and miR-328-3p decreased in both NNK-induced A/J mice and MLE-12 cells. Lico A could reverse the NNK-induced ectopic miRNA (miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p) expression both in vivo and in vitro and elicit in vivo lung cancer chemopreventive effect against NNK. In MLE-12 cells, the overexpression of miR-144-3p elicited the same effect as NNK regarding the expression of lung cancer biomarker CK19; the silencing of miR-144-3p reversed the effect of NNK on cell cycle distribution and apoptosis. Lico A could reverse the effect of NNK on the expression of miR-144-3p, CK19, and RASA1 (predicted target of miR-144-3p).ConclusionThe present study suggests that miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p were involved in the in vivo pathogenesis of NNK-induced lung cancer, and lico A could reverse the effect of NNK both in vivo and in vitro to elicit lung cancer chemopreventive effects through, at least partially, these five ectopically expressed miRNAs, especially miR-144-3p.     

Protein kinases involved in mating and osmotic stress in the yeast Kluyveromyces lactis

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Gα and Gβ, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.     

Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast

Background

The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/C^Ama1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.

Findings

Here we show that Ama1p is a substrate of APC/C^Cdc20 but not APC/C^Cdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/C^Cdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/C^Ama1-mediated Cdc20p degradation.

Conclusions

Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.

     

A comparison of maximal squat strength and 5-, 10-, and 20-meter sprint times, in athletes and recreationally trained men

The purpose of this study was to identify whether there was a relationship between relative strength during a 1 repetition maximum (1RM) back squat and 5-, 10-, and 20-m sprint performances in both trained athletes and recreationally trained individuals. Professional rugby league players (n = 24) and recreationally trained individuals (n = 20) participated in this investigation. Twenty-meter sprint time and 1RM back squat strength, using free weights, were assessed on different days. There were no significant (p ≥ 0.05) differences between the well-trained and recreationally trained groups for 5-m sprint times. In contrast, the well-trained group's 10- and 20-m sprint times were significantly quicker (p = 0.004; p = 0.002) (1.78 + 0.06 seconds; 3.03 + 0.09 seconds) compared with the recreationally trained group (1.84 + 0.07 seconds; 3.13 + 0.11 seconds). The athletes were significantly stronger (170.63 + 21.43 kg) than the recreationally trained individuals (135.45 + 30.07 kg) (p = 0.01); however, there were no significant differences (p > 0.05) in relative strength between groups (1.78 + 0.27 kg/kg; 1.78 + 0.33 kg/kg, respectively). Significant negative correlations were found between 5-m sprint time and relative squat strength (r = -0.613, power = 0.96, p = 0.004) and between relative squat strength and 10- and 20-m sprint times in the recreationally trained group (r = -0.621, power = 0.51, p = 0.003; r = -0.604, power = 0.53, p = 0.005, respectively). These results, indicating that relative strength, are important for initial sprint acceleration in all athletes but more strongly related to sprint performance over greater distances in recreationally trained individuals.     

CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65

The E3 ubiquitin ligase complex CDC20‐activated anaphase‐promoting complex/Cyclosome (APC/C^CDC20) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co‐activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well‐known cell cycle‐related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA‐binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11‐dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle‐independent function of CDC20, establishes APC11^CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone‐related diseases.     

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.     

The effect of post-lunch napping on mood,reaction time,and antioxidant defense during repeated sprint exercice

To compare the effects of two nap opportunities (20 and 90 min) to countermeasure the transient naturally occurring increased sleepiness and decreased performances during the post-lunch dip (PLD). Fourteen highly trained judokas completed in a counterbalanced and randomized order three test sessions (control (No-nap), 20- (N20) and 90-min (N90) nap opportunities). Test sessions consisted of the running-based anaerobic sprint test (RAST), simple and multiple-choice reaction times (MCRT) and the Epworth sleepiness scale (ESS). From the RAST, the maximum (P_max), mean (P_mean) and minimum (P_min) powers were calculated. Blood samples were taken before and after the RAST to measure the effect of pre-exercise napping on energetic and muscle damage biomarkers and antioxidant defense. N20 increased P_max and P_mean compared to No-nap (p < 0.001, d = 0.59; d = 0.66) and N90 (p < 0.001, d = 0.98; d = 0.72), respectively. Besides, plasma lactate and creatinine increased only when the exercise was performed after N20. Both N20 (p < 0.001, d = 1.18) and N90 (p < 0.01, d = 0.78) enhanced post-exercise superoxide dismutase activity compared to No-nap. However, only N20 enhanced post-exercise glutathione peroxidase activity (p < 0.001, d = 1.01) compared to pre-nap. Further, MCRT performance was higher after N20 compared to No-nap and N90 (p < 0.001, d = 1.15; d = 0.81, respectively). Subjective sleepiness was lower after N20 compared to No-nap (p < 0.05, d = 0.92) and N90 (p < 0.01, d = 0.89). The opportunity to nap for 20 min in the PLD enhanced RAST, MCRT performances, and antioxidant defense, and decreased sleepiness. However, the opportunity of 90 min nap was associated with decreased repeated sprint performances and increased sleepiness, probably because of the sleep inertia.     

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.     

Trisomy 20p: case report and genetic review

Partial trisomy for the distal part of the short arm of chromosome 20 reported in a girl aged 11/2 years with typical craniofacial dysmorphies and psychomotor retardation. The trisomy resulted from a paternal translocation t(14;20) (q32.3;p11.1). The review of 25 cases of partial trisomy 20p showed that most cases (22 : 25) were due to parental translocations. Predominant involvement of small chromosomes in translocations with chromosome 20 was also detected.     

Specificity and Versatility of Substrate Binding Sites in Four Catalytic Domains of Human N-Terminal Acetyltransferases

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate = MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate = MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate’s backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1’ sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.