Hot off the Press

In the Hot off the Press section of Molecular BioSystems members of the Editorial Board and their research groups highlight recent literature for the benefit of the community. This month the highlighted topics include the role of dopamine and parkin in Parkinson's disease, the use of a synthetic tryptophan metabolite in autoimmune diseases, and chitosan quantum dots as bioprobes.

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Chitosan quantum dots as bioprobes

Quantum dots (QD) continue to attract the attention of the scientific community. Because of their unique properties, they are used as efficient fluorescent probes and in several recent studies showed great promise in a range of biological applications. Water soluble QDs, as well as those modified with a variety of biomolecules such as DNA, proteins and small molecules have been successfully synthesized. Ongoing research is focused on improving cellular uptake and selective intracellular targeting.

A group of Chinese scientists have recently reported on the construction of polysaccharide labelled CdSe/ZnS QDs which can be used as bioprobes. They used chitosan and carboxymethyl chitosan polysasharides to prepare modified QDs by two different methods. One was based on simple grinding of CdSe/Zns QD in an agate mortar containing polysaccharide powder and the other method took advantage of the heavy metal chelating ability of carboxyl containing chitosan (Fig. 1). Both types of QD, average diameter of 5 nm, were water soluble and penetrated the membrane upon the incubation with live yeast cells. Additionally, the confocal images of the cells showed that they remained well dispersed and fluorescent in intracellular space. In contrast to mercaptoacetic acid containing QDs, chitosan QDs were not toxic most probably due to the chitosan ability to chelate Cd²⁺ which prevents its release. Since chitin is one of the components of the yeast cell wall, endocytosis helps the chitosan QDs' to penetrate the cell. Therefore, it could be possible to use this simple method to label cell organelles as well as to study drug release. This will be a focus of the authors' future research.

Fig. 1 Reproduced from Chem. Commun., 2005, 5518, by permission of The Royal Society of Chemistry.

 

M. Xie, H. H. Liu, P. Chen, Z. L. Zhang, X. H. Wang, Z. X. Xie, Y.M. Du, B.Q. Pan, D. W. Pang, Chem. Commun., 2005, 5518–5520.

Reviewed by: Ljiljana Fruk, Universitat Dortmund, Germany

Lipids changing shape

For intracellular trafficking, small membrane vesicles that are connected to a donor membrane through a thin tubular neck are formed. The formation of such highly curved membrane vesicles requires a significant amount of energy, and may ultimately lead to fission. One way to form and/or stabilize regions with a particular curvature, is to alter directly the shape of lipids constituting the membrane. This shape can be changed from an inverted-cone shape to a cone shape through the addition of an acyl chain by lysophospatidic acid acyl transferase (LPAAT) activity. Interestingly, such LPAAT activity has been reported for several proteins that are important for trafficking and fission (notably endophilin and CtBP/BARS). However, Harvey McMahon and coworkers claim that this activity is an artefact of the protein purification. Their research suggests that the LPAAT activity might be a consequence of a co-purification of contaminants due to the affinity that the proteins of interest have for membranes present in the bacteria used for purification. Whether direct modification of the shape of lipids by proteins plays a direct role in locally modulating membrane curvature in vivo is thus still a matter of active debate.

 

Jennifer L. Gallop, P. Jonathan G. Butler, Harvey T. McMahon, Nature, 2005, 438, 675–678.

Reviewed by: Gerbrand Koster (Lab. PhysicoChimie Curie UMR168) and Jean-Baptiste Manneville (UMR144), Institut Curie, Paris, France

Treating autoimmune diseases

Autoimmune disease afflicts millions of people in the developed world, and there remains a need for improved therapeutics for these diseases. In a recent issue of Science, Platten, et al. report the efficacy of an orally bioavailable compound, 3,4-DAA, in a mouse model for multiple sclerosis.

This study began with genome-wide expression analysis in a T cell line to show the effect of certain modified peptides that had been previously found to help induce self-tolerance to autoantigens and thus to treat autoimmune diseases. These modified peptides are not necessarily of pharmaceutical interest themselves due to their non-optimal pharmacokinetic properties, but it was reasoned that if one could understand how they work, drug targets for more practical compounds might be identified. A striking result from these studies was that expression of the indoleamine 2,3-dioxygenase (IDO) gene was stimulated over 70-fold upon treatment with the modified peptide. IDO is an enzyme involved in the catabolism of the amino acid tryptophan (Trp). This was very interesting because it was already known that degradation products of Trp play a role in modulating immune response (Fig. 2), with certain catabolites mediating an immunosuppressive effect by inducing apoptosis of activated T cells and dampening allogenic proliferation. This connection between the effects of the peptides and Trp catabolism led the investigators to examine the effects of different Trp catabolites on mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). All of the compounds were beneficial. Most striking was the efficacy of the aforementioned 3,4-DAA, an orally available synthetic mimic of Trp metabolites. They found that this compound reversed many of the symptoms of EAE in mice. A battery of in vivo and in vitro tests showed that this compound indeed suppressed the activity of autoreactive TH1 cells. Thus, treatment of human MS patients with 3,4-DAA or other Trp catabolite mimetics may be a viable strategy for the treatment of MS and other autoimmune diseases.

Fig. 2 Reprinted with permission from Science, 2005, 310, 850–855. Copyright 2005 AAAS.

 

M. Platten, P. P. Ho, S. Youssef, P. Fontoura, H. Garren, E. M. Hur, R. Gupta, L. Y. Lee, B. A. Kidd, W. H. Robinson, et al., Science, 2005, 310, 850–855.

Reviewed by: Thomas Kodadek, Departments of Internal Medicine and Molecular Biology and the Division of Translational Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-9185, USA.

Parkin modification and Parkinson's disease

The involvement of dopamine in the development of Parkinson's disease has been well documented, although the detailed mechanism is still unknown. The general scheme is that dopamine undergoes oxidation to form dopamine quinone, which reacts with functional groups on proteins to form covalent adducts that presumably contribute to disease progression. A recent study from researchers at the Harvard Medical School has shed light on how this process plays out. They have revealed that parkin, an important protein associated with inherited forms of Parkinson's disease, is vulnerable to dopamine modification.

Using in vitro methods, LaVoie and co-workers showed convincingly that parkin aggregates upon treatment with dopamine under oxidizing conditions. In one particularly interesting experiment, they used amino-phenyl boronate-modified agarose resin to pull down dopamine-modified proteins in cell lysates by virtue of the highly specific and stable interaction between catechols and boronic acids. They have also shown that dopamine modification of parkin caused significant loss of its E3 ubiquitin ligase activity. Based on the fact that parkin contains a large number of cysteine residues, the authors proposed that formation of the covalent adduct involved thiol addition to the quinone intermediate via a Michael addition-like mechanism.

In more difficult experiments on living cells, LaVoie, et al. demonstrated that both exogenous and endogenous dopamine stimulated the production of insoluble parkin monomer and aggregates. Moreover, studies on human Parkinson brains showed accumulated insoluble parkin monomer and aggregates. Thus, the dopamine-induced structural and functional modifications of parkin may be relevant to the intrinsic vulnerability of dopaminergic neurons under stress.

Dopamine-induced protein cross-linking has been demonstrated as a general event. Besides cysteine, histidine and lysine have also been implicated as reaction partners for the quinone intermediate under physiological conditions. It seems likely that many other physiologically relevant quinone-based modifications of proteins remain to be discovered.

 

Matthew J. LaVoie, Beth L. Ostaszewski, Andreas Weihofen, Michael G. Schlossmacher, and Dennis J. Selkoe, Nat. Med., 2005, 11(11), 1214.

Reviewed by: Bo Liu, Division for Translational Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Encapsulated photosensitizer for eradicating tumors

Finding efficient ways to eradicate tumor cells is the most important task in winning the battle against cancer. Although there are a number of drugs used in cancer treatment, several problems still remain such as efficient delivery to the infected cells and selective targeting. Photosensitizers are molecules which are capable of producing singlet oxygen when irradiated with light and can cause irreversible damage to the cell. They are therefore suitable candidates for anti-cancer drugs. However, their use can lead to significant side effects. Delivery of photosenzitiser molecules to ensure high local concentration and constant singlet oxygen quantum yields is still a problem.

A group of researchers from Harvard Medical School and the University of Connecticut tackled these problems by designing polymer nanoparticles containing photosensitizer molecules (Fig. 3). They used a chlorin derivative meso-tetraphenylporpholactol (TPP) as a photosensitizer because of its high singlet oxygen quantum yield and high light absorption. Additionally, the polarity of the molecule was ideal for encapsulation into a nanoparticle shell. The molecules aggregate in the nanoparticle core leading to chromaphore quenching. This is important because the molecule is then unable to fluoresce or induce phototoxicity whilst it is being transported. Poly (lactic-co-glycolic acid) (PLGA) was used as a nanoparticle matrix and TPP containing nanoparticles prepared via a solvent-diffusion method. The resulting particles had an average diameter of around 98 nm and were stable in the dark and at room temperature for 6–12 months. Upon treatment with 0.5% lipid solution, PLGA dissolved leading to the release of photosensitizer and restoration of fluorescence intensity. Interestingly, this was not observed with the control particles containing the photosensitizer porphyrin. The authors suggest that this is caused by difference in polarity between two molecules which makes the release of TPP more favourable. After different cell studies were carried out to test the nanoparticle toxicity, a set of experiments was performed in vivo using mice with prostate carcinoma cells. The mice were injected with nanoparticles and treated with light therapy. Within 3 days a reduction in tumor size was observed leading to the complete disappearance of the tumor by day 27. This final study showed that polymer nanoparticles encapsulating photosensitizer are extremely promising anticancer therapeutics. They are non-toxic in extracellular space; their delivery and intracellular release can be controlled and the used polymer is biodegradable which makes it attractive for clinical applications. There is a need for further characterisation and investigation of their potential, and further work concerning these issues is currently under way.

Fig. 3 Reprinted with permission from NanoLett., 2005, 5(12), 2552–2556. Copyright 2005 American Chemical Society.

 

J. R. McCarthy, J. Manuel Perez, C. Bruckner, R. Weissledder, NanoLett, 2005, 5(12), 2552–2556.

Reviewed by: Ljiljana Fruk, Universitat Dortmund, Germany

More ways to combat bacterial infections

Emerging antibiotic resistance is a significant public health issue, especially multidrug-resistant bacterial infections. None of the fourteen known classes of antibiotics targeting various aspects of bacterial macromolecule biosyntheses has escaped the resistance mechanism. Therefore, an important problem is to explore anti-bacterials with novel mechanisms of action. In this context, Heike Brötz-Oesterelt et al. reinvestigated a group of acydepsipeptides (ADEPs) originally isolated from Streptococcus hawaiiensis NRRL 15010.

ADEP1 and two other synthetic derivatives showed potent antibacterial activity both in vitro and in vivo against a wide range of Gram-positive multidrug-resistant strains. Biosynthetic assays using radiolabeled precursors to the major classes of macromolecules demonstrated that ADEPs do not function by any of the classical mechanisms involving inhibition of protein or nucleic acid synthesis, indicating a novel mechanism of bactericidal activity. In order to trace the biological target of ADEPs, a genetic approach was employed by transforming ADEP-sensitive E. coli with the genomic library of an ADEP-resistant E. coli strain which was selected in an ADEP-containing agar plate. By sequencing the plasmid carried in the newly ADEP-resistant E. coli, they found all of them to bear a point mutation in the gene encoding Clp protease (ClpP), a bacterial analogue of the 20S core particle of the eukaryotic proteasome. ClpP is a multimer that forms a barrel-like structure, the interior of which contains proteolytic active sites responsible for a great deal of programmed protein degradation in bacteria. This activity is manifested in concert with specialized ATPases that sit atop the ClpP barrel that act to bind and unwind substrates targeted for destruction. To verify that ADEPs interact with ClpP directly, two ADEP derivatives were synthesized that allowed their interaction with ClpP to be assessed by cross-linking and affinity binding experiments. Both of these methods demonstrated a direct interaction of ADEPs with ClpP.

Surprisingly, the authors further discovered that treatment with ADEPs resulted in the activation of the proteolytic activity of ClpP in vitro, even in the absence of the otherwise essential ATPases. This unusual proteolytic activation was also seen inside the bacterial cells by examining the whole proteome profile after treatment of ADEPs, where significant induction of protein degradation was seen. This is the first demonstration that small molecules can activate the proteolytic activity of ClpP allosterically. This result in toxicity to the bacteria is by presumably relaxing the tight regulation of ClpP-mediated proteolysis. This study has revealed a completely novel mechanism of action of an anti-bacterial compound.

 

Heike Brötz-Oesterhelt, Dieter Beyer, Hein-Peter Kroll, Rainer Endermann, Christoph Ladel, Werner Schroeder, Berthold Hinzen, Siegfried Raddatz, Holger Paulsen, Kerstin Henninger, Julia E Bandow, Hans-Georg Sahl, and Harald Labischinski, Nat. Med. 2005, 11(10), 1082–1087.

Reviewed by: Xiangshu Xiao, UT-Southwestern Medical Center, USA

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