Structure of a ternary complex of proteinase K,mercury, and a substrate-analogue hexa-peptide at 2.2 Å resolution

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH₂ has been determined at 2.2 Å resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 Sγ which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide. © 1996 Wiley-Liss, Inc.     

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Crystallization and structure determination of goat lactoferrin at 4.0 A resolution: a new form of packing in lactoferrins with a high solvent content in crystals

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO3(2-) ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with a=104.6 A, b=153.8 A, c=155.1 A and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 A resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 A3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 A resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO3(2-). ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.     

Mutation Spectrum of RB1 Gene in Unilateral Retinoblastoma Cases from Tunisia and Correlations with Clinical Features

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.     

HLA Class I and Class II Alleles and Haplotypes Confirm the Berber Origin of the Present Day Tunisian Population

In view of its distinct geographical location and relatively small area, Tunisia witnessed the presence of many civilizations and ethnic groups throughout history, thereby questioning the origin of present-day Tunisian population. We investigated HLA class I and class II gene profiles in Tunisians, and compared this profile with those of Mediterranean and Sub-Sahara African populations. A total of 376 unrelated Tunisian individuals of both genders were genotyped for HLA class I (A, B) and class II (DRB1, DQB1), using reverse dot-blot hybridization (PCR-SSO) method. Statistical analysis was performed using Arlequin software. Phylogenetic trees were constructed by DISPAN software, and correspondence analysis was carried out by VISTA software. One hundred fifty-three HLA alleles were identified in the studied sample, which comprised 41, 50, 40 and 22 alleles at HLA-A,-B,-DRB1 and -DQB1 loci, respectively. The most frequent alleles were HLA-A*02:01 (16.76%), HLA-B*44:02/03 (17.82%), HLA-DRB1*07:01 (19.02%), and HLA-DQB1*03:01 (17.95%). Four-locus haplotype analysis identified HLA-A*02:01-B*50:01-DRB1*07:01-DQB1*02:02 (2.2%) as the common haplotype in Tunisians. Compared to other nearby populations, Tunisians appear to be genetically related to Western Mediterranean population, in particular North Africans and Berbers. In conclusion, HLA genotype results indicate that Tunisians are related to present-day North Africans, Berbers and to Iberians, but not to Eastern Arabs (Palestinians, Jordanians and Lebanese). This suggests that the genetic contribution of Arab invasion of 7^th-11^th century A.D. had little impact of the North African gene pool.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A,and their combination in cultured Vero cells

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food‐borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl‐2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose‐dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl‐2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:42–50, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20310