Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis

The synthesis of novel fluorogenic retro-aldol substrates for aldolase antibody 38C2 is described. These substrates are efficiently and specifically processed by antibody aldolases but not by natural cellular enzymes. Together, the fluorogenic substrates and antibody aldolases provide reporter gene systems that are compatible with living cells. The broad scope of the antibody aldolase allows for the processing of a range of substrates that can be designed to allow fluorescence monitoring at a variety of wavelengths. We also have developed the following concept in fluorescent protein tags. β-Diketones bearing a fluorescent tag are bound covalently by the aldolase antibody and not other proteins. We anticipate that proteins fused with the antibody can be tagged specifically and covalently within living cells with fluorophores of virtually any color, thereby providing an alternative to green fluorescent protein fusions.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 102-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

Clusters of charged residues in protein three-dimensional structures.

Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Unravelling metabolism of Leishmania by metabolomics

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.