Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.     

Complex Structure of Engineered Modular Domains Defining Molecular Interaction between ICAM-1 and Integrin LFA-1

Intermolecular contacts between integrin LFA-1 (α(L)β(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.     

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser?Pulses

Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.     

A PH domain within OCRL bridges clathrin‐mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.     

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA)² (2–6), where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7, 8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction “hub” according to their binding affinity.Quantitative estimation of protein-protein interactions in vivo will require the amount of the complex to be directly measured or the level of reconstituted reporters to be directly proportional to the strength of the interactions. To achieve quantitative analysis of protein interactions in eukaryotic expression system, we have designed a yeast surface two-hybrid (YS2H) system that can express a pair of proteins, one protein as a fusion to a yeast cell wall protein, agglutinin, and the other in a secretory form. When two proteins interact in this system, they associate in the secretory pathway, and the prey that would otherwise be released into the media is captured on the surface by the bait. We have devised two different schemes to quantitatively estimate the affinity of two interacting molecules: flow cytometric detection of antibody binding to the epitope tags fused to the prey and the bait, and the GFP readout from the complementation of split GFP fragments fused to the prey and the bait. They are induced under a bi-directional promoter to promote a synchronized and comparable level of expression.Herein we demonstrate the quantitative nature of YS2H in predicting the affinity between two interacting proteins, particularly in the range of 100 pm to 10 μm. This feature allowed us to examine specific interactions between antigen and antibody, to identify hot spots of allosteric activation in integrins, and to isolate camelid heavy chain-only antibodies against botulinum neurotoxin as components of therapeutic agents to treat botulism (9). With the incorporation of PCA technique into the YS2H, our system may be developed into an in vivo tool to measure the kinetics of protein-protein interactions. Potential applications of YS2H include affinity maturation of antibodies, differentiation of weak to high affinity binders to the hub protein in interaction networks, and confirmation of hypothetical interacting pairs of proteins in a high throughput manner.     

Chip-based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen

The development of reagents with high affinity and specificity to the antigens of hepatitis C virus (HCV) is important for the early stage diagnosis of its infection. Aptamers are short, single-stranded oligonucleotides with the ability to specifically recognize target molecules with high affinity. Herein, we report the selection of RNA aptamers that bind to the core antigen of HCV. High affinity aptamers were isolated from a 10(15) random library of 60 mer RNAs using the SELEX procedure. Importantly, the selected aptamers specifically bound to the core antigen, but not to another HCV antigen, NS5, in a protein chip-based assay. Using these aptamers, we developed an aptamer-based biosensor for HCV diagnosis and detected the core antigen from HCV infected patients' sera with good specificity. This novel aptamer-based antigen detection sensor could be applied to the early diagnosis of HCV infection.     

Comparative microbiome analysis of Haemaphysalis longicornis ticks at the Korea Combat Training Center in 2022

The Korea Combat Training Center (KCTC), located in Gangwon Province, is a restricted military training facility where research on the environmental conditions and health risks to military personnel has been limited. In this study, using iSeq 100, we investigated the bacterial abundance and microbiome of Haemaphysalis longicornis specimens collected at the KCTC from June to August 2022, to assess current and potential public health risks to military personnel. Our results show that adult ticks had significantly greater species richness compared with larvae and nymphs, with no notable differences in diversity across developmental stages. Principal coordinate analysis of the microbial communities did not show differences attributable to any single factor, such as collection location or date. Coxiella-like endosymbionts (AB001519) were identified in all 13 samples, and Jatrophihabitans, Sphingomonas, and Spirosoma were consistently found across all samples. In addition, iSeq 100 also identified Rickettsia rickettsii and Borrelia spp., which were not detected with conventional polymerase chain reaction (PCR).     

Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice

We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community.