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In eukaryotic cells, mitochondria play a key role in apoptosis; however, ancestral eukaryotic cells such as Giardia lamblia only possess a mitochondrial remnant, the mitosome. Interestingly, this protozoan still undergoes an apoptosis‐like process; therefore, we focused primarily on the search for the mitochondria‐independent executor DNase. Here, we identified, cloned, expressed, and characterized the caspase‐activated DNase (CAD) from Giardia lamblia . Using a commercial polyclonal antibody that recognizes mouse, rat, and human caspase‐activated DNase (hCAD), we developed an immunoproteomic analysis using a crude extract of curcumin‐treated Giardia lamblia trophozoites (CEGl) and detected a spot of 42 kDa and pI 9.4, similar to hCAD and sequenced by LC‐MS. The proteomic profile matched a novel protein of 383 residues, with a predicted 42 kDa, pI 9.4, a CIDE‐N domain, and putative H‐K‐H catalytic motif. Afterward, we cloned the full‐length gene (GenBank: ON707040), expressed it, and purified it as a 6‐His tag‐recombinant protein in Escherichia coli , which was also recognized by commercial anti‐CAD. In conclusion, genetic, proteomic, and structural analyses showed that the identified gCAD is an orthologous protein of hCAD, and its DNase role in the apoptosis‐like signaling pathway of Giardia lamblia can be further analyzed.

Hemophilia A, one of the most common genetic bleeding disorders, is caused by various mutations in the blood coagulation factor VIII (F8) gene. Among the genotypes that result in hemophilia A, two different types of chromosomal inversions that involve a portion of the F8 gene are most frequent, accounting for almost half of all severe hemophilia A cases. In this study, we used a transcription activator-like effector nuclease (TALEN) pair to invert a 140-kbp chromosomal segment that spans the portion of the F8 gene in human induced pluripotent stem cells (iPSCs) to create a hemophilia A model cell line. In addition, we reverted the inverted segment back to its normal orientation in the hemophilia model iPSCs using the same TALEN pair. Importantly, we detected the F8 mRNA in cells derived from the reverted iPSCs lines, but not in those derived from the clones with the inverted segment. Thus, we showed that TALENs can be used both for creating disease models associated with chromosomal rearrangements in iPSCs and for correcting genetic defects caused by chromosomal inversions. This strategy provides an iPSC-based novel therapeutic option for the treatment of hemophilia A and other genetic diseases caused by chromosomal inversions.

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

Gene targeting by homologous recombination is a powerful method to manipulate the genome precisely and could be exploited to correct genetic defects. Zinc finger nucleases are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease. Zinc finger nucleases were generated that stimulated gene targeting from half-site sequences from the human beta-globin gene and the human common gamma-chain gene. Zinc finger nucleases were also generated that stimulated gene targeting at full sites from the green fluorescent protein gene and the human CD8alpha gene. This work built on the prior zinc finger design work of others and in targeting these four genes had a 100% success rate at designing nucleases to the consensus half-site 5'-GNNGNNGNN-3' and the consensus full site 5'-NNCNNCNNCNNNNNNGNNGNNGNN-3', suggesting that zinc finger nucleases can be empirically designed to stimulate gene targeting in a large portion of the mammalian genome.

The prolonged persistence of extracellular chromatin and DNA is a salient feature of diseases like cystic fibrosis, systemic lupus erythematosus and COVID-19 associated microangiopathy. Since deoxyribonuclease I (DNase1) is a major endonuclease involved in DNA-related waste disposal, recombinant DNase1 is an important therapeutic biologic. Recently we described the production of recombinant murine DNase1 (rmDNase1) in Pichia pastoris by employing the α-mating factor prepro signal peptide (αMF-SP) a method, which we now applied to express recombinant human DNASE1 (rhDNASE1). In addition to an impaired cleavage of the αMF pro-peptide, which we also detected previously for mDNase1, expression of hDNASE1 resulted in a 70–80 times lower yield although both orthologues share a high structural and functional homology. Using mDNase1 expression as a guideline, we were able to increase the yield of hDNASE1 fourfold by optimizing parameters like nutrients, cultivation temperature, methanol supply, and codon usage. In addition, post-translational import into the rough endoplasmic reticulum (rER) was changed to co-translational import by employing the signal peptide (SP) of the α-subunit of the Oligosaccharyltransferase complex (Ost1) from Saccharomyces cerevisiae . These improvements resulted in the purification of ~ 8 mg pure mature rmDNase1 and ~ 0.4 mg rhDNASE1 per Liter expression medium of a culture with a cell density of OD 600 = 40 in 24 hours. As a main cause for the expression difference, we assume varying folding abilities to reach a native conformation, which induce an elevated unproductive unfolded protein response within the rER during hDNASE1 expression. Concerning functionality, rhDNASE1 expressed in P. pastoris is comparable to Pulmozyme®, i.e. rhDNASE1 produced in Chinese hamster ovary (CHO) cells by Roche - Genentech. With respect to the biochemical effectivity, rmDNase1 is superior to rhDNASE1 due to its higher specific activity in the presence of Ca 2 + /Mg 2 + and the lower inhibition by monomeric actin.

Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris . Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

The hypothetical protein ‘Alr3200’ of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62×10 4 Kunitz Units (KU) mg −1 protein. Circular dichroism analysis predicted Alr3200 to have ~40% β-strands and ~9% α-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, An alr3200 + , the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of γ-radiation, which is the LD 50 for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se , but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena , which hampers DNA repair resulting in radiosensitivity.

The programmed senescence of flower petals has been shown to involve the fragmentation of nuclear DNA. Nuclear DNA fragmentation, as determined by the TUNEL assay, was detected in Petunia×hybrida corollas during both pollination-induced and age-related senescence. DNA fragmentation was detected late in the lifespan of the flower when corollas were wilting and producing ethylene. The induction of a 43 kDa nuclease (PhNUC1) correlated with increased DNA fragmentation. PhNUC1 is a glycoprotein with activity against DNA and RNA and a pH optimum of 7.5. EDTA was found to inhibit PhNUC1 activity, but the addition of Co2+ restored activity in the presence of the chelating agent. When total protein extracts from senescing petals were fractionated by differential centrifugation, PhNUC1 activity was detected in the nuclear but not the cytoplasmic fraction. Activity of PhNUC1 was induced in non-senescing corollas by treatment with ethylene. Delayed increases in PhNUC1 activity observed in ethylene-insensitive flowers (35S:etr1-1) suggest that ethylene modulates the timing of PhNUC1 induction, but that it is not an absolute requirement for its activation.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB+/SpeA−/Sda1low phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB+/SpeA−/Sda1high phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2- component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (ΔCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.