Custom-designed zinc finger nucleases: What is next?

Custom-designed zinc finger nucleases (ZFNs)--proteins designed to cut at specific DNA sequences--combine the non-specific cleavage domain (N) of Fok I restriction endonuclease with zinc finger proteins (ZFPs). Because the recognition specificities of the ZFPs can be easily manipulated experimentally, ZFNs offer a general way to deliver a targeted site-specific double-strand break (DSB) to the genome. They have become powerful tools for enhancing gene targeting--the process of replacing a gene within a genome of cells via homologous recombination (HR)--by several orders of magnitude. ZFN-mediated gene targeting thus confers molecular biologists with the ability to site-specifically and permanently alter not only plant and mammalian genomes but also many other organisms by stimulating HR via a targeted genomic DSB. Site-specific engineering of the plant and mammalian genome in cells so far has been hindered by the low frequency of HR. In ZFN-mediated gene targeting, this is circumvented by using designer ZFNs to cut at the desired chromosomal locus inside the cells. The DNA break is then patched up using the new investigator-provided genetic information and the cells' own repair machinery. The accuracy and high efficiency of the HR process combined with the ability to design ZFNs that target most DNA sequences (if not all) makes ZFN technology not only a powerful research tool for site-specific manipulation of the plant and mammalian genomes, but also potentially for human therapeutics in the future, in particular for targeted engineering of the human genome of clinically transplantable stem cells.     

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.     

RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence

The double-stranded RNA binding domain (dsRBD) is a small protein domain of 65–70 amino acids adopting an αβββα fold, whose central property is to bind to double-stranded RNA (dsRNA). This domain is present in proteins implicated in many aspects of cellular life, including antiviral response, RNA editing, RNA processing, RNA transport and, last but not least, RNA silencing. Even though proteins containing dsRBDs can bind to very specific dsRNA targets in vivo, the binding of dsRBDs to dsRNA is commonly believed to be shape-dependent rather than sequence-specific. Interestingly, recent structural information on dsRNA recognition by dsRBDs opens the possibility that this domain performs a direct readout of RNA sequence in the minor groove, allowing a global reconsideration of the principles describing dsRNA recognition by dsRBDs. We review in this article the current structural and molecular knowledge on dsRBDs, emphasizing the intricate relationship between the amino acid sequence, the structure of the domain and its RNA recognition capacity. We especially focus on the molecular determinants of dsRNA recognition and describe how sequence discrimination can be achieved by this type of domain.     

Über Struktur und Aktivität der den H2O2-Zerfall katalysierenden Cu2+-Komplexe. — VI. Differenzierung von nativer RNS und nativer DNS bzw. denaturierter DNS auf Grund der katalytischen Eigenschaften

Summary The catalysis of H₂O₂ decomposition by Cu²⁺-complexes of RNA and DNA has been investigated. It is shown that both complexes decompose H₂O₂, but only the Cu²⁺-RNA-system shows peroxidative activity too, e.g. only in this case the nucleotide bases are degraded. Thermal denaturation of DNA also leads to a Cu²⁺-complex with peroxidative activity, the latter being dependent on the degree of denaturation.

---

---

V. Mitteilung:S. Petri, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 1778 (1966).

---

12. Mitteilung überMetallionen und H ₂ O ₂; 11. Mitteilung:H. Ch. Curtius, P. Anders, R. Zell, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 2256 (1966).     

The current structural and functional understanding of APOBEC deaminases

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases has emerged as an intensively studied field as a result of their important biological functions. These enzymes are involved in lipid metabolism, antibody diversification, and the inhibition of retrotransposons, retroviruses, and some DNA viruses. The APOBEC proteins function in these roles by deaminating single-stranded (ss) DNA or RNA. There are two high-resolution crystal structures available for the APOBEC family, Apo2 and the C-terminal catalytic domain (CD2) of Apo3G or Apo3G-CD2 [Holden et al. (Nature 456:121–124, 2008); Prochnow et al. (Nature 445:447–451, 2007)]. Additionally, the structure of Apo3G-CD2 has also been determined using NMR [Chen et al. (Nature 452:116–119, 2008); Furukawa et al. (EMBO J 28:440–451, 2009); Harjes et al. (J Mol Biol, 2009)]. A detailed structural analysis of the APOBEC proteins and a comparison to other zinc-coordinating deaminases can facilitate our understanding of how APOBEC proteins bind nucleic acids, recognize substrates, and form oligomers. Here, we review the recent development of structural and functional studies that apply to Apo3G as well as the APOBEC deaminase family.     

Aptamer and its applications in neurodegenerative diseases

Aptamers are small single-stranded DNA or RNA oligonucleotide fragments or small peptides, which can bind to targets by high affinity and specificity. Because aptamers are specific, non-immunogenic and non-toxic, they are ideal materials for clinical applications. Neurodegenerative disorders are ravaging the lives of patients. Even though the mechanism of these diseases is still elusive, they are mainly characterized by the accumulation of misfolded proteins in the central nervous system. So it is essential to develop potential measures to slow down or prevent the onset of these diseases. With the advancements of the technologies, aptamers have opened up new areas in this research field. Aptamers could bind with these related target proteins to interrupt their accumulation, subsequently blocking or preventing the process of neurodegenerative diseases. This review presents recent advances in the aptamer generation and its merits and limitations, with emphasis on its applications in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathy, Huntington’s disease and multiple sclerosis.     

CAP2, cyclase-associated protein 2, is a dual compartment protein

Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.