Jobst Landgrebe

The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes.

Recently, the human C(alpha)-formylglycine (FGly)-generating enzyme (FGE), whose deficiency causes the autosomal-recessively transmitted lysosomal storage disease multiple sulfatase deficiency (MSD), has been identified. In sulfatases, FGE posttranslationally converts a cysteine residue to FGly, which is part of the catalytic site and is essential for sulfatase activity. FGE is encoded by the sulfatase modifying factor 1 (SUMF1) gene, which defines a new gene family comprising orthologs from prokaryotes to higher eukaryotes. The genomes of E. coli, S. cerevisiae and C. elegans lack SUMF1, indicating a phylogenetic gap and the existence of an alternative FGly-generating system. The genomes of vertebrates including mouse, man and pufferfish contain a sulfatase modifying factor 2 (SUMF2) gene encoding an FGE paralog of unknown function. SUMF2 evolved from a single exon SUMF1 gene as found in diptera prior to divergent intron acquisition. In several prokaryotic genomes, the SUMF1 gene is cotranscribed with genes encoding sulfatases which require FGly modification. The FGE protein contains a single domain that is made up of three highly conserved subdomains spaced by nonconserved sequences of variable lengths. The similarity among the eukaryotic FGE orthologs varies between 72% and 100% for the three subdomains and is highest for the C-terminal subdomain, which is a hotspot for mutations in MSD patients.

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein‐2 (LAMP‐2) and LAMP‐1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP‐1 and LAMP‐2 (LAMP−/−), low‐density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild‐type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP‐2 deficient liver and that overexpression of LAMP‐2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP‐2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP−/− cells was diminished by overexpression of any of the three isoforms of LAMP‐2, but not by LAMP‐1. The LAMP‐2 luminal domain, the membrane‐proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP‐2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone‐mediated autophagy function of LAMP‐2.

Efficient design and analysis of two colour factorial microarray experiments

Statistical microarray data analysis has evolved fast in the last 5 years. However, design issues related to gene expression profiling experiments using two-colour cDNA or oligonucleotide-based microarrays have been investigated only recently. Different approaches and criteria for the choice of efficient designs have been proposed, which apply to a wide range of experiments from simple to complex factorial experimental setups. Here, results from previous research are expanded by showing how contrast vectors and matrices describing the experimental questions under investigation can be used to tailor efficient and economic designs that take into account the estimability of the desired contrasts. The most efficient design among a set of candidate designs is found by applying the e-efficiency criterion from optimal design theory suitable for general factorial designs, which reduces to the comparison of variance factors in the case of vectorial contrasts. These results enable researchers to select efficient designs among several candidate designs for arbitrary complex factorial designs given a set of experimental questions. The corresponding algorithms are implemented in the open source R package daMA available at www.bioconductor.org. The electronic version of this paper can be found at www.sciencedirect.com.

Lysosomal Proteome and Transcriptome

As reflected in this monograph, a lot of knowledge about the lysosomal compartment has been acquired since the discovery of the organelle by de Duve and others in the 1950s. Nevertheless, a lot of questions are still open, and some of them can be addressed using screening techniques. Among them are questions about the structure of the lysosomal mem-brane, the function of its proteins, and the effect of lysosomal dysfunction on cell functions. Although the proteins of the lysosomal matrix are well known, little is realised about the proteins of the lysosomal membrane. In the first section of this chapter, we describe the most important proteome analysis related techniques used to purify and analyse lysosomal structures that can help to define the complete set of matrix proteins and to describe the pro-teins of the lysosomal membrane. In the second section, we delineate the gene expression pro-filing technique and briefly describe approaches which can be used to analyse the influence of lysosomal dysfunctions on the cell, as well as on the function of lysosomal proteins. Because transcriptome and proteome analyses of lysosomal structure and function are still only in the initial stage, we will focus on the description of the methods and the major aims.

Active Contours and a Background Intensity Estimator for Analysis of Microarray Spots

Differential gene expression experiments using DNA microarray technology yield raw data in the form of fluorescence images which must be segmented and quantified to extract the expression data.