Chia-Lin Chyan

Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence.

Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles.

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Aβ peptides

Aggregated β‐amyloid (Aβ) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a β‐sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Aβ sequence remains unclear. To explore the correlation between Aβ sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full‐length and fragment‐truncated Aβ peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C‐terminus, particularly the amino acids of 17–21, 25–35, and 41–42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17–21, 25–35 or 41–42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22–24 or residues 36–40 had little effect on toxicity and aggregative ability. While the N‐terminal residues 1–16 may not play a major role in neurotoxicity and aggregation, a lack of N‐terminal fragment Aβ peptide, (e.g. Aβ17–35), does not display the neurotoxicity of either full‐length or 17–21, 25–35 truncated Aβ peptides.

α-Glycosylation by d-Glucosamine-Derived Donors: Synthesis of Heparosan and Heparin Analogues That Interact with Mycobacterial Heparin-Binding Hemagglutinin

Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted β-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.

The effects of macromolecular crowding on the mechanical stability of protein molecules.

Macromolecular crowding, a common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins. A single‐molecule method based on atomic force microscopy (AFM) was used to investigate the effects of macromolecular crowding on the forces required to unfold individual protein molecules. It was found that the mechanical stability of ubiquitin molecules was enhanced by macromolecular crowding from added dextran molecules. The average unfolding force increased from 210 pN in the absence of dextran to 234 pN in the presence of 300 g/L dextran at a pulling speed of 0.25 μm/sec. A theoretical model, accounting for the effects of macromolecular crowding on the native and transition states of the protein molecule by applying the scaled‐particle theory, was used to quantitatively explain the crowding‐induced increase in the unfolding force. The experimental results and interpretation presented could have wide implications for the many proteins that experience mechanical stresses and perform mechanical functions in the crowded environment of the cell.

Cloning and expression of a seed-specific metallothionein-like protein from sesame

A cDNA clone, SiMT encoding an Ec type of metallothionein (MT)-like protein, was isolated from maturing seeds of sesame (Sesamum indicum L.), and its deduced protein sequence shared 47–65% similarity to other known Ec type of MT-like proteins with three highly conserved cysteine-rich segments. The transcript of SiMT was exclusively accumulated in maturing seeds from two weeks after flowering to the end of seed maturation. The results of a southern blot analysis suggested that one SiMT and one SiMT-like gene were present in the sesame genome. Recombinant SiMT fused with glutathione-S-transferase (GST) was over-expressed in Escherichia coli, and purified to homogeneity by affinity chromatography. Recombinant SiMT released from GST was harvested after factor Xa cleavage. Migration of the recombinant SiMT during SDS–PAGE was accelerated when its binding metal ions were depleted by EDTA. The metal-binding capability of recombinant SiMT was measured by inductively-coupled plasma atomic emission spectrometry. Our results show that the recombinant SiMT could trap zinc or copper ions, but not manganese ions, with a stoichiometric ratio (metal ion/SiMT) of approximately 2.

Stability of Artificial Oil Bodies constituted with Recombinant Caleosins

Caleosin is a unique calcium binding protein anchoring to the surface of seed oil bodies by its central hydrophobic domain composed of an amphiphatic alpha-helix and a proline-knot subdomain. Stable artificial oil bodies were successfully constituted with recombinant caleosin overexpressed in Escherichia coli. The stability of artificial oil bodies was slightly or severely reduced when the amphiphatic alpha-helix or proline-knot subdomain in the hydrophobic domain of caleosin was truncated. Deletion of the entire central hydrophobic domain substantially increased the solubility of the recombinant caleosin, leading to a complete loss of its capability to stabilize these oil bodies. A recombinant protein engineered with the hydrophobic domain of caleosin replaced by that of oleosin, the abundant structural protein of seed oil bodies, could stabilize the artificial oil bodies, in terms of thermo- and structural stability, as effectively as caleosin or oleosin.

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable Ki values of 1.18×10−10 M and 9.53×10−11 M, respectively. The recombinant cystatins were found to be thermally stable up to 60 °C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.

Solution Structure of a Phytocystatin from Ananas comosus and Its Molecular Interaction with Papain

The structure of a recombinant pineapple cystatin (AcCYS) was determined by NMR with the RMSD of backbone and heavy atoms of twenty lowest energy structures of 0.56 and 1.11 Å, respectively. It reveals an unstructured N-terminal extension and a compact inhibitory domain comprising a four-stranded antiparallel β-sheet wrapped around a central α-helix. The three structural motifs (G45, Q89XVXG, and W120) putatively responsible for the interaction with papain-like proteases are located in one side of AcCYS. Significant chemical shift perturbations in two loop regions, residues 45 to 48 (GIYD) and residues 89 to 91 (QVV), of AcCYS strongly suggest their involvement in the binding to papain, consistent with studies on other members of the cystatin family. However, the highly conserved W120 appears not to be involved in the binding with papain as no chemical shift perturbation was observed. Chemical shift index analysis further indicates that the length of the α-helix is shortened upon association with papain. Collectively, our data suggest that AcCYS undergoes local secondary structural rearrangements when papain is brought into close contact. A molecular model of AcCYS/papain complex is proposed to illustrate the interaction between AcCYS and papain, indicating a complete blockade of the catalytic triad by AcCYS.

Characterization of Oil Bodies in Adlay (Coix lachryma-jobi L)

Oil bodies were observed in cells of both embryo and aleurone layers of mature adlay grains (Coix lachryma-jobi L. var. ma-yuen Stapf). Stable oil bodies were successfully isolated from the adlay grains. Thin-layer chromatography revealed that the contents stored in the adlay oil bodies were mainly neutral lipids (>90% triacylglycerols and about 5% diacylglycerols). The integrity of the isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms (termed oleosin-H and oleosin-L) and one caleosin were present in the adlay oil bodies. Full-length cDNA fragments encoding these three unique oil-body proteins were obtained by PCR cloning. MALDI-MS analyses confirmed that the three full-length cDNA fragments encoded the two oleosin isoforms and one caleosin observed in the oil bodies isolated from the adlay grains.

The effect of PKA-phosphorylation on the structure of inhibitor-1 studied by NMR spectroscopy

Inhibitor-1 is an acid- and heat-stable protein. It can be turned into a potent inhibitor of protein phosphatase-1 (PP1) after phosphorylation at Thr35 by c-AMP-dependent protein kinase (PKA). Although it has been known that pre-phosphorylation is essential for inhibition of PP1, the structure-function relationship of Thr(35)-phosphorylated inhibitor-1, such as whether or not PKA-phosphorylation pre-triggers conformational changes in inhibitor-1, remains unclear. In this study, we performed structural characterization of Thr(35)-phosphoroylated inhibitor-1 by using multi-dimensional heternuclear NMR spectroscopy. The result of structural comparison between Thr(35)-phosphoroylated and non-phosphorylated inhibitor-1 indicated that PKA-phosphorylation has no significant effect on the global conformation of free-state inhibitor-1. This finding may support the inference that regulation of the interactions between inhibitor-1 and PP1 through PKA-phosphorylation mainly depends on the phosphate group instead of phosphorylation-induced conformational change.