Polyhistidine Affinity Chromatography: From the Idea to a Method of Broad Applicability and the Pitfalls in-between.

The present article recapitulates the development of the polyhistidine affinity tag purification principle. Emphasis is laid on events behind the scenes which were never published. The key concept of the method emerged in a team discussion and its further development was driven by the need of Roche in-house projects.

Determination of free desmosine in human plasma and its application in two experimental medicine studies.

Elastin is one of the major extracellular matrix proteins associated with connective tissue. Its degradation leads to the liberation of the unique amino acids desmosine and isodesmosine. These have shown utility as biomarkers of elastin breakdown for disease progression, patient stratification, and drug efficacy. So far, the quantitation of desmosines in plasma is hampered by complex sample preparation. Here we demonstrate an improved and simplified procedure for detecting both free and total desmosines. The method is based on spiking with a deuterium-labeled desmosine standard, ethanol precipitation, propionylation, high-performance liquid chromatography (HPLC) separation, and selected reaction monitoring (SRM) mass spectrometry. The performance of the assay is illustrated by comparing the levels of free and total desmosines in normal healthy plasma and those from patients diagnosed with chronic obstructive pulmonary disease (COPD). A conserved ratio of 1:3 for free to total desmosine was found. The determination of free desmosine has higher accuracy than that of total desmosine; therefore, it is the method of choice when plasma volume is limiting. Finally, we show that the plasma desmosine concentration correlates with age and body mass index.

Bace2 is a ß cell-enriched protease that regulates pancreatic ß cell function and mass.

Decreased ß cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human ß cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented ß cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of ß cell maintenance and provide a rational strategy to inhibit this protease for the expansion of functional pancreatic ß cell mass.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans.

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

3D structure of Alzheimer's amyloid-beta(1-42) fibrils.

Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-beta (Abeta) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Abeta(1-42), which was obtained by using hydrogen-bonding constraints from quenched hydrogen/deuterium-exchange NMR, side-chain packing constraints from pairwise mutagenesis studies, and parallel, in-register beta-sheet arrangement from previous solid-state NMR studies. Although residues 1-17 are disordered, residues 18-42 form a beta-strand-turn-beta-strand motif that contains two intermolecular, parallel, in-register beta-sheets that are formed by residues 18-26 (beta1) and 31-42 (beta2). At least two molecules of Abeta(1-42) are required to achieve the repeating structure of a protofilament. Intermolecular side-chain contacts are formed between the odd-numbered residues of strand beta1 of the nth molecule and the even-numbered residues of strand beta2 of the (n - 1)th molecule. This interaction pattern leads to partially unpaired beta-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Abeta fibril growth. It also provides a structural basis for fibrillization inhibitors.

Protection of rat primary hippocampal cultures from A beta cytotoxicity by pro-inflammatory molecules is mediated by astrocytes.

The brain of Alzheimer's disease patients shows abundant dystrophic neurites in close proximity to fibrillar beta-amyloid (A beta) plaques, and activated glial cells. We evaluated the influence of pro-inflammatory molecules (LPS + IFN-gamma) on A beta(1-42) neurotoxicity. 2 microM A beta(1-42) induced apoptosis of hippocampal cells and LPS + IFN-gamma reduced the apoptosis induced by A beta. However, LPS + IFN-gamma prevented apoptosis only in hippocampal cultures containing astrocytes. Also, LPS + IFN-gamma induced the secretion of TGF beta, a cytokine having neuroprotective effects, only in hippocampal cultures that contained astrocytes. Astrocytes had a regulatory effect over microglial and neuronal responses to A beta. The results suggest that LPS + IFN-gamma, traditionally considered as pro-apoptotic, reduced apoptosis induced by A beta through the activation of neuroprotective mechanisms mediated by astrocytes. We propose that astrocytes are pivotal in the modulation of inflammation of the CNS. The impairment of the regulatory functions performed by activated astrocytes could represent an important pathogenic mechanism for neurodegenerative diseases.

Quantification of the A beta peptide in Alzheimer's plaques by laser dissection microscopy combined with mass spectrometry.

The accumulation and aggregation of the beta-amyloid peptide (A beta) in the brain represents a key factor in the pathogenesis of Alzheimer's disease (AD). Many of the transgenic mouse models for AD exhibit an amyloid pathology with neuritic plaques but they typically vary by the type and abundance of plaques identified in their brains and by the onset and severity of cognitive impairment. Thus, an important consideration in the characterization of AD transgenic mouse models should be the quantitative evaluation of the amyloid load in the brain together with a detailed physico-chemical analysis of A beta from the deposited plaques. Here we present an analytical procedure to collect single amyloid plaques from anatomically defined brain regions by laser dissection microscopy that can be quantitatively assessed in their A beta isoforms composition by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Quantification was achieved by stable isotope dilution using calibrated 15N-labeled A beta standards that were spiked in the sample immediately after laser dissection. Using this method, we found that the amyloid loads in brain plaques isolated from the transgenic AD mouse model PS2APP or from human were similar. Total A beta composition was estimated at approximately 50-100 fmol per excised plaque disc, as confirmed by immunoblot analysis. N-Terminal truncated A beta isoforms were identified in both transgene and human amyloid plaques but with significantly elevated levels in human samples.

Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases.

The full-length and ectodomain forms of beta-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the beta-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2'0 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM/DAEFR, K(m) = 7 microm, K(cat) = 0.002 s(-1); Swedish mutant, SEVNL/DAEFR, K(m) = 9 microm, K(cat) = 0.02 s(-1)). A new substrate (VVEVDA/AVTP, K(m) = 1 microm, K(cat) = 0.004) was identified by serendipity.