◆　Restriction enzyme HindIII

◆　Intermediate Filaments

◆ Antimicrobial and Antifungal Peptides

◆　　Restriction enzyme HindIII

Restriction enzyme HindIII is a type II restriction endonuclease which specifically digests palindromic sequence AAGCTT. The gene of this enzyme was cloned into a vector so that we could get and purify large amounts of the enzyme from Escherichia coli host. Several kinds of mutant proteins were obtained by protein engineering methods, and their enzymic properties were examined (see Fig.1). Among them E86K, has a two-fold higher activity than the wild one, showing altered substrate specificity in the presence of Mn⁺⁺ or Co⁺⁺. Now three dimensional structure of HindIII is under investigation. We have been collaborating with Dr. N. Watanabe, Hokkaido University for its determination. Crystallization of this enzyme a hard work. The wild type structure and its derivatives will surely be elucidated by the use of Sincrotoron Radiation Apparatus which will be built this year,in Tosu City, Saga. As a reference, structure of a restriction enzyme BamHI is illustrated in Fig. 2.

Ｆｉgure1

◆　 Intermediate Filaments

Intermediate filaments (IFs) are a major component of the cytoskeleton in higher eukaryotic cells. They appear to play an important role in providing mechanical integrity to cells and are critically involved in cell division, motility and other cellular processes. IF proteins are comprised of a large family of gene products, and are commonly capable of polymerization into IFs with a diameter of ~10 nm. From the extent of sequence homology and the pattern of cell type specific expression, IF proteins are classified into at least five different types: type I, acidic keratins; type II, neutral-basic keratins; type III, vimentin, desmin and glial fibrillary acidic protein; type IV, three neurofilament proteins; type V, lamins. IF proteins share a characteristic tripartite structure that includes the central a-helical rod domain flanked by the non-a-helical N-terminal head and C-terminal tail domains. Although the head and tail domains are of variable lengths, sequences and chemical characteristics, the rod domain consisting of ~310 amino acids (~350 for lamins), has long heptad-repeats and relatively conserved sequences at the N- and C-terminal ends.

Assembly of IFs from the subunit proteins is a multi-step process and is not fully understood. An object of our studies is to clarify the molecular mechanism that governs the assembly of IFs, focusing on the specific roles of the three domains of IF proteins. We prepare recombinant IF proteins and the related fragments to characterize their abilities to form IFs and the domain-domain interactions required for IF assembly. These studies would provide useful information to understand how the head domain-specific phosphorylation by several protein kinases induces disassembly of IFs. Furthermore, these studies would help to establish causal relationships between mutations of IF protein genes and human diseases.

Figure2

◆ Antimicrobial and Antifungal Peptides

Antimicrobial and antifungal peptides are now recognized as a critical first line of defense against many pathogens. We are trying to find new peptides from unique organisms in Saga area, to study the expression systems and the structure-function relationships and also to exploit as useful medicines.