Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bil... more Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bilayer, which surrounds the approximately 400 A diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The E1-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.
To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modifi... more To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modified to display breast cancer cell targeting functional group (LXY30). Five surface-exposed residues were mutated to cysteine to allow conjugation to maleimide-linked chemical groups via thiol-selective linkages. Engineered virus-like nanoparticles were then covalently conjugated to a breast cancer recognized ligand, LXY30 and an amine-coupled near-infrared fluorescence dye. LXY30-HEV VLP was checked for its binding and entry to a breast cancer cell line and for tumor targeting in vivo to breast cancer tissue in mice. The engineered virus-like nanoparticle not only targeted cancer cells, but also appeared immune silent to native hepatitis E virus antibodies due to epitope disruption at the antibody-binding site. These results demonstrate the production of a theranostic capsule suitable for cancer diagnostics and therapeutics based on surface modification of a highly stable virus-like nanoparticle.
Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. S... more Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.
Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development... more Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development, complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C and novel antigenic properties have been described for C Envs. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an acutely infected (Fiebig stage I/II) subject was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane proximal external region (MPER), extended by a poly-lysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan-blade" motifs when visualized by cryo-electron microscopy. The CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16 and VRC01 HIV-1 neutralizing monoclonal antibodies (mAbs), as well as the V1/V2 specific PGT121, 697, 2158 and 2297 mAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimer...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2014
Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, ... more Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolong...
ABSTRACT This review covers only a fraction of the area of one written a dozen years ago on photo... more ABSTRACT This review covers only a fraction of the area of one written a dozen years ago on photosynthesis in cyanobacteria (Ho and Krogmann, 1982) yet it cites many more references than that earlier work. The power of reductionist laboratory science has increased immensely in the intervening years. The soluble electron transfer catalysts of photosynthesis have received disproportionate attention since soluble proteins are more easily dealt with by the techniques of protein chemistry. Research on each of the catalysts reviewed here has exposed a variety of insights through the tools of contemporary science. The many studies of different forms of ferredoxin in cyanobacteria promise new understanding of the regulation of electron transfer and its mechanism. The crystal structures of ferredoxin, ferredoxin-NADP+ oxidoreductase, flavodoxin and plastocyanin are elegant examples of what our broader understanding will become. The powerful technique of gene deletion used on cytochrome c6 (cytochrome c553) has done more than confirm preconceptions. It has given us the intriguing puzzle of why more than two routes of electron flow between carriers of very similar redox potential may be used. The low potential cytochrome c beckons for an explanation of its catalytic function and for an understanding of its role in the ancient origin of other cytochromes. Finally, hydrogenase, whose catalytic act is the simplest—the movement of an electron to or from a proton—seems ready for understanding. Hydrogenase has a long history of fragility and activity loss during purification. One type of hydrogenase has now been purified and there are glimpses of its metabolic role.
Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bil... more Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bilayer, which surrounds the approximately 400 A diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The E1-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.
To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modifi... more To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modified to display breast cancer cell targeting functional group (LXY30). Five surface-exposed residues were mutated to cysteine to allow conjugation to maleimide-linked chemical groups via thiol-selective linkages. Engineered virus-like nanoparticles were then covalently conjugated to a breast cancer recognized ligand, LXY30 and an amine-coupled near-infrared fluorescence dye. LXY30-HEV VLP was checked for its binding and entry to a breast cancer cell line and for tumor targeting in vivo to breast cancer tissue in mice. The engineered virus-like nanoparticle not only targeted cancer cells, but also appeared immune silent to native hepatitis E virus antibodies due to epitope disruption at the antibody-binding site. These results demonstrate the production of a theranostic capsule suitable for cancer diagnostics and therapeutics based on surface modification of a highly stable virus-like nanoparticle.
Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. S... more Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.
Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development... more Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development, complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C and novel antigenic properties have been described for C Envs. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an acutely infected (Fiebig stage I/II) subject was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane proximal external region (MPER), extended by a poly-lysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan-blade" motifs when visualized by cryo-electron microscopy. The CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16 and VRC01 HIV-1 neutralizing monoclonal antibodies (mAbs), as well as the V1/V2 specific PGT121, 697, 2158 and 2297 mAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimer...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2014
Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, ... more Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolong...
ABSTRACT This review covers only a fraction of the area of one written a dozen years ago on photo... more ABSTRACT This review covers only a fraction of the area of one written a dozen years ago on photosynthesis in cyanobacteria (Ho and Krogmann, 1982) yet it cites many more references than that earlier work. The power of reductionist laboratory science has increased immensely in the intervening years. The soluble electron transfer catalysts of photosynthesis have received disproportionate attention since soluble proteins are more easily dealt with by the techniques of protein chemistry. Research on each of the catalysts reviewed here has exposed a variety of insights through the tools of contemporary science. The many studies of different forms of ferredoxin in cyanobacteria promise new understanding of the regulation of electron transfer and its mechanism. The crystal structures of ferredoxin, ferredoxin-NADP+ oxidoreductase, flavodoxin and plastocyanin are elegant examples of what our broader understanding will become. The powerful technique of gene deletion used on cytochrome c6 (cytochrome c553) has done more than confirm preconceptions. It has given us the intriguing puzzle of why more than two routes of electron flow between carriers of very similar redox potential may be used. The low potential cytochrome c beckons for an explanation of its catalytic function and for an understanding of its role in the ancient origin of other cytochromes. Finally, hydrogenase, whose catalytic act is the simplest—the movement of an electron to or from a proton—seems ready for understanding. Hydrogenase has a long history of fragility and activity loss during purification. One type of hydrogenase has now been purified and there are glimpses of its metabolic role.
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