Stefan Kammerer

Pex19p, a Farnesylated Protein Essential for Peroxisome Biogenesis

ABSTRACT We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.

Association testing by DNA pooling: An effective initial screen

With an ever-increasing resource of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. We provide a proof of concept of the use of pooled DNA as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall. We have examined 15 SNPs in the cholesteryl ester transfer protein (CETP) gene, a gene previously demonstrated to be associated with serum high-density lipoprotein cholesterol levels. The SNPs were amplified in two pools of DNA derived from groups of individuals with extremely high and extremely low serum high-density lipoprotein cholesterol levels, respectively. P values <0.05 were obtained for 14 SNPs, supporting the described association. Genotyping of the individual samples showed that the average margin of error in frequency estimate was ≈4% when pools were used. These findings clearly demonstrate the potential of pooling techniques and their associated technologies as an initial screen in the search for genetic associations.

Designing isoform-specific peptide disruptors of protein kinase A localization

A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (KD = 1–2 nM) and high specificity for RIα versus RIIα. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIα-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.

Identification of the semaphorin receptor PLXNA2 as a candidate for susceptibility to schizophrenia

The discovery of genetic factors that contribute to schizophrenia susceptibility is a key challenge in understanding the etiology of this disease. Here, we report the identification of a novel schizophrenia candidate gene on chromosome 1q32, plexin A2 (PLXNA2), in a genome-wide association study using 320 patients with schizophrenia of European descent and 325 matched controls. Over 25 000 single-nucleotide polymorphisms (SNPs) located within approximately 14 000 genes were tested. Out of 62 markers found to be associated with disease status, the most consistent finding was observed for a candidate locus on chromosome 1q32. The marker SNP rs752016 showed suggestive association with schizophrenia (odds ratio (OR)=1.49, P=0.006). This result was confirmed in an independent case–control sample of European Americans (combined OR=1.38, P=0.035) and similar genetic effects were observed in smaller subsets of Latin Americans (OR=1.26) and Asian Americans (OR=1.37). Supporting evidence was also obtained from two family-based collections, one of which reached statistical significance (OR=2.2, P=0.02). High-density SNP mapping showed that the region of association spans approximately 60 kb of the PLXNA2 gene. Eight out of 14 SNPs genotyped showed statistically significant differences between cases and controls. These results are in accordance with previous genetic findings that identified chromosome 1q32 as a candidate region for schizophrenia. PLXNA2 is a member of the transmembrane semaphorin receptor family that is involved in axonal guidance during development and may modulate neuronal plasticity and regeneration. The PLXNA2 ligand semaphorin 3A has been shown to be upregulated in the cerebellum of individuals with schizophrenia. These observations, together with the genetic results, make PLXNA2 a likely candidate for the 1q32 schizophrenia susceptibility locus.

Large-Scale Association Study Identifies ICAM Gene Region as Breast and Prostate Cancer Susceptibility Locus

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

Cloning and characterization of the gene encoding the human peroxisomal assembly protein Pex3p.

Proteins essential for the assembly of functional peroxisomes are designated peroxins and are encoded by PEX genes. In yeast, Pex3p was previously identified as a peroxisomal integral membrane protein indispensable for peroxisome biogenesis and integrity. Here we report the cloning of the orthologous human PEX3 gene. It encodes a polypeptide of 373 amino acids (42 kDa) and is expressed in all tissues examined. As shown by transfection of epitope tagged constructs and immunofluorescence analysis, human Pex3p is localized at the peroxisome. The N‐terminal 40 amino acids were revealed to be sufficient to target a GFP reporter protein to the peroxisome. A positively charged five amino acid sequence within this N‐terminal region is highly conserved from yeast to human Pex3p. Overexpression of human Pex3p leads to proliferation of ER membranes in COS7 cells. Since disruption of human peroxins has been shown to result in peroxisomal biogenesis disorders, PEX3 is another candidate gene being involved in this disease group.

Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: A disease susceptibility polymorphism

The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimers disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity and/or mortality. To test this assumption, we compared allele frequencies of 6,500 single-nucleotide polymorphisms (SNPs) located in ≈5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (d-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an independent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. The Ile/Val SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound ≈3-fold weaker to the protein kinase A (PKA)-RIα isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of the recombinantly expressed PKA-RIα isoform. Our study suggests that alterations in PKA-RIα subcellular localization caused by variation in d-AKAP2 may have a negative health prognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health.

Polymorphisms in the gene for the human B2-bradykinin receptor. New tools in assessing a genetic risk for bradykinin-associated diseases

The B2-bradykinin receptor gene has been proposed as one of the candidate genes involved in the complex genetic underpinnings of common chronic disorders such as hypertension, ischemic heart disease or allergic asthma. Suitable genetic markers are needed to study these hypotheses. Therefore, it was our aim to identify polymorphic sites in the B2-receptor gene. Up to now, we characterized four polymorphisms: one in the promoter region and three other ones in each of the exons. Possible biological consequences are delineated and preliminary results of allele specific different biological action are shown.

Suppression of RAD21 gene expression decreases cell growth and enhances cytotoxicity of etoposide and bleomycin in human breast cancer cells

A genome-wide case-control association study done in our laboratory has identified a single nucleotide polymorphism located in RAD21 as being significantly associated with breast cancer susceptibility. RAD21 is believed to function in sister chromatid alignment as part of the cohesin complex and also in double-strand break (DSB) repair. Following our initial finding, expression studies revealed a 1.25- to 2.5-fold increased expression of this gene in several human breast cancer cell lines as compared with normal breast tissue. To determine whether suppression of RAD21 expression influences cellular proliferation, RNA interference technology was used in breast cancer cell lines MCF-7 and T-47D. Proliferation of cells treated with RAD21-specific small inhibitory RNA (siRNA) was significantly reduced as compared with mock-transfected cells and cells transfected with a control siRNA (Lamin A/C). This inhibition of proliferation correlated with a significant reduction in the expression of RAD21 mRNA and with an increased level of apoptosis. Moreover, MCF-7 cell sensitivity to two DNA-damaging chemotherapeutic agents, etoposide and bleomycin, was increased after inhibition of RAD21 expression with a dose reduction factor 50 (DRF50) of 1.42 and 3.71, respectively. At the highest concentrations of etoposide and bleomycin administered, cells transfected with a single siRNA duplex targeted against RAD21 showed 57% and 60% survival as compared with control cells, respectively. Based on these findings, we conclude that RAD21 is a novel target for developing cancer therapeutics that can potentially enhance the antitumor activity of chemotherapeutic agents acting via induction of DNA damage.

Symptoms in carriers of adrenoleukodystrophy relate to skewed X inactivation

Skewing of X inactivation may contribute to the manifestation of symptoms in adrenoleukodystrophy carriers. We observed highly skewed X inactivation in 32% of 22 symptomatic and asymptomatic carriers but not in 7 related and 35 unrelated controls. Skewing of X inactivation correlated with clinical neurological scores but not with the extent of very long‐chain fatty acid accumulation. Transcript analysis in cultured fibroblasts revealed that skewing could occur both in favor of the mutant and the wild‐type allele. Adrenoleukodystrophy carriers are more susceptible to develop skewing of X inactivation in favor of the mutant allele beiing associated with the manifestation of symptoms.