Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter.

Peroxisomes: Organelles at the crossroads.

Recent years have seen remarkable progress in our understanding of the function of peroxisomes in higher and lower eukaryotes. Combined genetic and biochemical approaches have led to the identification of many genes required for the biogenesis of this organelle. This review summarizes recent, rather surprising, results and discusses how they can be incorporated into the current view of peroxisome biogenesis.

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane.

Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling.

Pex5p, which is the import receptor for peroxisomal matrix proteins harboring a type I signal sequence (PTS1), is mono- and polyubiquitinated in Saccharomyces cerevisiae. We identified Pex5p as a molecular target for Pex4p-dependent monoubiquitination and demonstrated that either poly- or monoubiquitination of the receptor is required for the ATP-dependent release of the protein from the peroxisomal membrane to the cytosol as part of the receptor cycle. Therefore, the energy requirement of the peroxisomal import pathway has to be extended by a second ATP-dependent step, namely receptor monoubiquitination.

The peroxisomal protein import machinery.

Peroxisomes are unique organelles whose physiological functions vary depending on the cellular environment or metabolic and developmental state of the organism. These changes in enzyme content are accomplished by the dynamically operating membrane and matrix protein import machineries of peroxisomes that rely on the concerted function of at least 20 peroxins. The import of folded matrix proteins is mediated by cycling receptors that shuttle between the cytosol and peroxisomal lumen.

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels.

Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse.

DNA-based molecular wires: multiple emission pathways of individual constructs.

The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes.

Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation.

Generation of two-photon States with an arbitrary degree of entanglement via nonlinear crystal superlattices.

We demonstrate a general method of engineering the joint quantum state of photon pairs produced in spontaneous parametric down-conversion. The method makes use of a superlattice structure of nonlinear and linear materials, in conjunction with a broadband pump, to manipulate the group delays of the signal and idler photons relative to the pump pulse, and realizes photon pairs described by a joint spectral amplitude with arbitrary degree of entanglement. This method of group-delay engineering has the potential of synthesizing a broad range of states including factorizable states crucial for quantum networking and states optimized for Hong-Ou-Mandel interferometry.

Peroxisomal matrix protein receptor ubiquitination and recycling.

The peroxisomal targeting signal type1 (PTS1) receptor Pex5 is required for the peroxisomal targeting of most matrix proteins. Pex5 recognises target proteins in the cytosol and directs them to the peroxisomal membrane where cargo is released into the matrix, and the receptor shuttles back to the cytosol. Recently, it has become evident that the membrane-bound Pex5 can be modified by mono- and polyubiquitination.

Potential of the Akt inhibitor LY294005 to antagonize the efficacy of Cisplatin against HCT116 tumor cells in a DNA mismatch repair-dependent manner.

Human colorectal adenocarcinoma sublines either deficient (HCT116+ch2) or proficient (HCT116+ch3) in the function of MLH1, one of five proteins crucial to DNA mismatch repair (MMR), were used to investigate whether the Akt-specific inhibitor LY294005 could not only increase the efficacy of platinum drugs in HCT116 cells in general but also increase the efficacy of the cisplatinum compounds Cisplatin and Lipoplatin specifically in MLH1-deficient, Cisplatin- and Lipoplatin-resistant HCT116 cells. We report that, under the conditions it increased the efficacy of Docetaxel and did not affect that of 6-thioguanine, LY294005 decreased the sensitivity of both sublines to Cisplatin, Lipoplatin, Oxaliplatin, and Lipoxal. Notably, the LY294005-imposed decrease was significantly higher in the MLH1-proficient than in the MLH1-deficient subline with Cisplatin and Lipoplatin, whereas it was nearly the same in both sublines with Oxaliplatin and Lipoxal.

Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers.

Functional association of the AAA complex and the peroxisomal importomer.

The AAA peroxins, Pex1p and Pex6p, are components of the peroxisomal protein import machinery required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We demonstrate that Pex1p and Pex6p form a stable complex in the cytosol, which associates at the peroxisomal membrane with their membrane anchor Pex15p and the peroxisomal importomer. The interconnection of Pex15p with the components of the importomer was independent of Pex1p and Pex6p, indicating that Pex15p is an incorporated component of the assembly.

Investigation of peroxisome biogenesis revealed possible new roles for autophagy components.

Precursor aminopeptidase I oligomerizes in the cytosol and is imported into the vacuole as a dodecamer via the cytoplasm-to-vacuole targeting (Cvt) pathway or autophagy. However, this is not the only example for the import of oligomeric protein complexes into an organelle. During peroxisome biogenesis folded and oligomeric proteins can be imported into the lumen of the organelle.

Membrane association of the cycling peroxisome import receptor Pex5p.

Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex.

Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites.

Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26.

Pex19p-dependent targeting of Pex17p, a peripheral component of the peroxisomal protein import machinery.

Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p.

Non-invasive detection of fluorescence from exogenous chromophores in the adult human brain.

This is the first report on results proving that fluorescence of exogenous dyes inside the human brain can be excited and detected non-invasively at the surface of the adult head. Boli of indocyanine green (ICG) were intravenously applied to healthy volunteers, and the passage of the contrast agent in the brain was monitored by detecting the corresponding fluorescence signal following pulsed laser excitation at 780 nm. Our hypothesis that the observed fluorescence signal contains a considerable cortical fraction was corroborated by performing measurements with picosecond temporal resolution and analyzing distributions of times of arrival of photons, hence taking advantage of the well-known depth selectivity of that method.

Spectral identification of specific photophysics of cy5 by means of ensemble and single molecule measurements.

The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g.

Topogenesis of peroxisomal proteins does not require a functional cytoplasm-to-vacuole transport.

Folded and even oligomeric proteins can be imported from the cytosol into vacuoles and into peroxisomes. Pro-aminopeptidase I (prAPI) oligomerizes into a dodecamer and is imported into the vacuole via the cytoplasm-to-vacuole transport (cvt) pathway. How peroxisomes accommodate folded proteins is completely unknown.

Dynamin-related proteins and Pex11 proteins in peroxisome division and proliferation.

The abundance and size of cellular organelles vary depending on the cell type and metabolic needs. Peroxisomes constitute a class of cellular organelles renowned for their ability to adapt to cellular and environmental conditions. Together with transcriptional regulators, two groups of peroxisomal proteins have a pronounced influence on peroxisome size and abundance.

Peroxisomal matrix protein import: the transient pore model.

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a translocation pore by the import receptor, the ubiquitylation of the import receptors, and pore disassembly/ receptor recycling.

Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol.

Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol.

Direct observation of delayed fluorescence from a remarkable back-isomerization in Cy5.

The direct observations of delayed fluorescence and phosphorescence from the cyanine dye Cy5 are reported. The delayed fluorescence is generated from the S(1) state of trans-Cy5 through a reserve intersystem crossing from the cis-triplet state T(1) to the trans-singlet state S(1) via thermal activation. The lowest cis-triplet state is evidenced to be involved in the formation of the isomer.

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins.

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530).