Preclinical Alterations in the Serum of COL(IV)A3(-)/(-) Mice as Early Biomarkers of Alport Syndrome.

The efficiency of the inhibition of the angiotensin converting enzyme, the most widely used therapy for the Alport syndrome, depends on the onset of the therapy-the earlier the better. Hence, early progressive biomarkers are urgently required to allow for preclinical diagnosis, an early start of possible therapy as well as the monitoring of this therapy. In the present study, an improved comprehensive and precise proteomic approach has been applied to the serum of juvenile Alport-mice, nontreated and treated, and wild-type controls of various ages to search for biomarkers. With a total of 2542 stringently altered proteins, the serum composition clearly shows a dependency on age, that is, stage, and therapy. Initially, the serum constituents indicate an enhanced extracellular matrix remodeling, cell damage, and the production of particular acute phase proteins. A panel of 15 potential biomarker candidates has been identified. In later stages, renal filtration failure and systemic acute phase reaction determine the composition of the serum; an effect that is well-known for manifested human Alport syndrome. With a small number of mouse urine samples, for example, the proteomic results for gelsolin could be verified using ELISA. Once verified in man, these early biomarkers would allow for a sensitive and specific diagnosis of the Alport syndrome in children as well as facilitate the monitoring of a possible therapy.

The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis.

Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tightly bound via its GPI-anchor to the gut membrane. A function in defense against gut microbes is discussed.

Structure and mechanism of iron translocation by a Dps protein from Microbacterium arborescens.

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.

A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications.

Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.

Rapid hydrolysis of quorum-sensing molecules in the gut of lepidopteran larvae.

Microorganisms compete for nutrients and living space in the gut of plant-feeding insect larvae, such as Spodoptera spp. Their physiological activities and their organization are generally controlled or synchronised by "autoinducers", such as N-acylhomoserinelactones (AHLs). Due to the strongly alkaline milieu in the insect gut, the lactone ring of AHLs is rapidly and spontaneously opened. Further degradation to the inactive components homoserine and the acyl moiety is then achieved by a microbial N-acylamino acid hydrolase (AAH) and related enzymatic activities in the insect gut. Initialised by the alkaline milieu, such activities might account for the complete absence of AHLs in the intestinal fluid of the studied Spodoptera spp. The AHL-recognition system of E. coli RV308pSB40, but not that of Agrobacterium tumefaciens NT1/pZLR4 and Chromobacterium violaceum CV026, was found to be inhibited by the structurally related N-acylglutamines, which are abundantly present in the gut of many lepidopteran larvae. Our observations suggest an active role of the insect in interfering with the quorum sensing of their gut microbiota by several independent strategies.

The intricacy of biomarker complexity-the identification of a genuine proteomic biomarker is more complicated than believed.

Several reasons have been put forward to explain the irreproducibility of proteomic biomarker search. However, these reasons pertain to almost every part of biomarker search across the entire analytical workflow but are entirely experimental or methodological. However, in this article we point out that there is a further cause of such irreproducibility. This is not an additional methodological or experimental cause but arises directly from the biology of protein expression. It arises from the fact that disease changes the diversity within protein families. This cause of irreproducibility has been very little studied in relation to proteomic biomarker search. Gene expression is highly variable even in healthy people. Therefore, multiple proteoforms are also to be expected when gene expression is disrupted by disease, proteoforms that may be differently altered by pathology. In consequence, it is illogical to expect that the whole protein family produces a reliably usable biomarker. It is more reasonable to expect that a specific proteoform fulfills this role. Appropriate sample pre-fractionation methods and data analyses could help to identify this version, carrying the modification or the epitope required.

Acute phase proteins as promising biomarkers: Perspectives and limitations for human and veterinary medicine.

Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favor the systemic regulation of defense, coagulation, proteolysis, and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the nonspecificity of the acute phase reaction. Moreover, it appears that various extrahepatic tissues are also able to express APPs. These extrahepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of hepatic and extrahepatic APP species can be expected in blood. Hence, a selective alteration of parts of APPs might be expected. There are several hints on multiple molecular forms and fragments of tissue-derived APPs. These differences offer the chance for multiple selective determinations. Thus, specific proteoforms might indeed serve as tissue-specific disease indicators.

A novel Dps-type protein from insect gut bacteria catalyses hydrolysis and synthesis of N-acyl amino acids.

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding. The isolated AAH catalyses the hydrolysis of the amide bond (K(m) = 36 micromol l(-1)) and, less efficient, the formation (K(m) = 3 mmol l(-1)) of the elicitor active N-acyl amino acids. The AAH from M. arborescens SE14 shows no homology to known fatty acyl amidases (EC 3.5.1.4) but belongs to the family of Dps proteins (DNA-binding protein from starved cell). In line with other DPS proteins AAH is a homododecamer (monomer 17 181 Da) and contains iron atoms (c. 1-16 iron atoms per subunit). Unlike genuine DPS proteins the enzyme does not significantly bind DNA. Amino acid hydrolase is the first member of the DPS family that catalyses the cleavage or formation of amide bonds. The participation of a microbial enzyme in the homeostasis of N-acyl-glutamines in the insect gut adds further complexity to the interaction between plants and their herbivores.

Quantification of invertase activity in ants under field conditions.

Invertases (EC 3.2.1.26) are hydrolases that cleave sucrose into the monosacccharides, glucose, and fructose. They play a central role in carbohydrate metabolism of plants and animals. Methods presented so far to quantify invertase activity in ants or other animals have been hampered by the variability in both substrates and products of the enzymatic reaction in animals whose carbohydrate metabolism is highly active. Our method is based on a spectrophotometric quantification of the kinetics of glucose release. We first obtained an equilibrium state summarizing reactions of any carbohydrates and enzymes that are present in the extract. Sucrose was then added to quantify invertase activity as newly released glucose. Invertase activities differed significantly among species of ants. Variances were lowest among individuals from the same colony and highest among different species. When preparations were made from ants of the same species, invertase activity was linearly related to the number of ants used for extraction. Our method does not require ants to be kept on specific substrates prior to the experiment, or expensive or large equipment. It, thus, appears suitable for dealing with a broad range of physiological, ecological, and evolutionary questions.