[Show abstract][Hide abstract] ABSTRACT: To complement an earlier analysis of protein alterations in plasma from uremic versus healthy subjects by addition of further LC-MS/MS analysis to the previously used MALDI-TOF mass analyses.
Sequence identifications of tryptic peptides from SDS gel electrophoretic fractions of immunodepleted and HPLC-fractionated plasma was performed from seven chronic kidney disease stage 5 patients (age 55 ± 14 years, glomerular filtration rate 6.9 ±2.9 mL/minute/1.73 m2) and from seven matched controls.
About twice as many proteins were increased in uremic plasma as the previously identified. The identifications included proteins that consistently complement the two identification patterns regarding separate subunits from the same protein complex.
Mass spectrometric analysis is applicable to complex plasma proteomes in clinical settings. The LC-MS/MS technique, based on individual peptide sequence analyses, gives increased identifications and also demonstrates feasibility of this technique in clinical practice.
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION:
Is it possible to derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine?
We developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components.
WHAT IS KNOWN ALREADY:
The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes.
STUDY DESIGN, SIZE, DURATION:
Human cadaveric testicular tissue was exposed for 24 or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulphate (SDS). Acellular samples were used for further scaffold characterization purposes.
PARTICIPANTS/MATERIALS, SETTING, METHODS:
The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. Retention of testicular tissue-specific characteristics was evaluated by mass spectrometry, immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds.
MAIN RESULTS AND THE ROLE OF CHANCE:
Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure and the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, using proteomic analysis, we revealed numerous additional ECM proteins in DTM, indicating its complex nature. The mass spectrometry data were deposited to the ProteomeXchange with identifier PXD001524. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay not showing an aberrant fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration.
LIMITATIONS, REASONS FOR CAUTION:
The functionality of human testicular cells in DTM needs to be investigated.
WIDER IMPLICATIONS OF THE FINDINGS:
Our results suggest that the insights into the molecular composition of the testicular ECM provide new clues for the unravelling of its important yet poorly understood role in regulating testicular function, and DTM-based bioscaffolds are promising components for the development of human in vitro spermatogenesis as a treatment for various types of male fertility disorders.
STUDY FUNDING/COMPETING INTERESTS:
This study is supported by a Ph.D. grant from the Agency for Innovation by Science and Technology (IWT) and research grants from the Flemish League Against Cancer-Public Utility Foundation (VLK), the Scientific Research Foundation Flanders (FWO), the Vrije Universiteit Brussel, the Swedish Research Council/ Finnish Academy of Science, Emil och Wera Cornells Stiftelse, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. J.-B.S. was supported by the German Research Foundation (DFG-Grant No.: STU 506/3-1). E.G. and J.D.K. are postdoctoral fellows of the Scientific Research Foundation Flanders. The authors declare that no competing interests exist.
TRIAL REGISTRATION NUMBER:
Full-text · Article · Nov 2014 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: It is now about half a century since molecular research on alcohol dehydrogenase (ADH), short-chain dehydrogenase/reductase (SDR) and medium-chain dehydrogenase/reductase (MDR) started. During this time, at least four stages of research can be distinguished, which led to many ADH, SDR and MDR structures from which their origins could be traced. An introductory summary of these stages is given, followed by a current update on the now known structures, including the present pattern of mammalian MDR-ADH enzymes into six classes and their evolutionary relationships. In spite of the wide spread in evolutionary changes from the “constant” class III to the more “variable” other classes, the change in class V (only confirmed as a transcript in humans) and class VI (absent in humans) are also restricted. Such spread in variability is visible also in other dehydrogenases, but not always so restricted in other co-evolving proteins we have studied. Finally, the shift in era of present ADH research is highlighted, as well as levels of likely future continuation.
No preview · Article · Oct 2014 · Chemico-Biological Interactions
[Show abstract][Hide abstract] ABSTRACT: Author Summary
The spinning process of spider silk is crucial for making webs or other complex constructions to catch spider's prey. The main components of the silk are spidroins, which are large and repetitive proteins that have conserved nonrepetitive terminal domains (NT and CT). Spiders manage both to store the highly aggregation-prone spidroins in solution at extreme concentrations in the silk glands and then to rapidly convert these spidroins into a solid fiber within fractions of a second as they spin fibres. This process has been extensively studied and is thought to involve a pH gradient, but how this pH gradient is generated and maintained was not resolved. Here, we measured the pH at locations along the ampullate gland and determined that the pH decreases to 5.7 in the middle of the spinning duct. We also observed that the carbon dioxide pressure is simultaneously increased and that its accumulation may affect the stability of CT. We find that active carbonic anhydrase (CA) is crucial to maintain the pH gradient along the gland. Detailed molecular studies of NT and CT under the disparate conditions present along the gland revealed a lock and trigger mechanism whereby in more neutral pH conditions, precocious spidroin aggregation is prevented, and when in more acidic pH conditions, NT dimers firmly interconnect the spidroins and the CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins. We conclude that this mechanism enables temporal and spatial control of silk formation and may be harnessed in attempts to produce artificial silk replicas.
[Show abstract][Hide abstract] ABSTRACT: The C-peptide of proinsulin exhibits multiple activities and several of the underlying molecular interactions are known. We recently showed that human C-peptide is sub-divided into a tripartite architecture and that the pattern, rather than the exact residue positions, is a characteristic feature. We have now analyzed 75 proinsulins, ranging from fish to human and find a limited co-evolution with insulin, but with many marked deviations. This suggests a complex relationship, in which not only insulin affects the evolution of C-peptide. A subdivided nature, however, is a characteristic feature among all C-peptides, with the N-terminal segment the one most conserved. This segment, ascribed chaperoning charge-interactions with insulin, suggests that the insulin interactions constitute a basic function, although largely shifting from Glu to Asp residues in C-peptides of lower life forms. A second conserved feature is a mid-segment with a high content of adjacent Pro and Gly residues, in mammalian C-peptides compatible with a turn structure, but with fewer and more distantly interspaced such residues in the non-mammalian forms, and even absent in several fish forms. However, this segment of coelacanth C-peptide possesses a unique Cys distribution, capable of forming a disulfide-stabilized turn. Finally, the C-terminal segment of mammalian C-peptides, ascribed a possible receptor-interacting function, is not really discernable in the sub-mammalian forms. Combined, these patterns suggest an evolutionary stepwise acquisition of the tripartite mammalian C-peptide molecule, with insulin-interaction being ancestral, various turn stabilizations apparently of intermediate emergence, and possible receptor-interaction the most recent addition.
Full-text · Article · Jul 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Abstract Aggregation of transthyretin (TTR), a plasma-binding protein for thyroxine and retinol-binding protein, is the cause of several amyloid diseases. Disease-associated mutations are well known, but wild-type TTR is, to a lesser extent, also amyloidogenic. Monomerization, not oligomer formation as in several other depository diseases, is the rate-limiting step in TTR aggregation, and stabilization of the natively tetrameric form can inhibit amyloid formation. Modifications on Cys10, as well as interactions with native ligands in plasma, were early found to influence the equilibrium between tetrameric and monomeric TTR by dissociating or stabilizing the tetramer. Following these discoveries, synthetic ligands for pharmacological prevention of TTR aggregation could be developed. In this article, we outline how the different types of TTR interactions and its microheterogeneity in plasma are related to its propensity to form amyloid fibrils. We conclude that plasma constituents and dietary components may act as natural TTR stabilizers whose mechanisms of action provide cues for the amelioration of TTR amyloid disease.
No preview · Article · Jun 2014 · Biomolecular concepts
[Show abstract][Hide abstract] ABSTRACT: Abstract Many biological roles have been assigned to proinsulin C-peptide over the years. Some appear surprisingly disparate and sometimes even contradictory, like chaperone-like actions and depository tendencies. This review summarizes recently reported biomolecular interactions of the peptide and presents how they correlate with structural and functional aspects into a partitioned molecular architecture. At the structural level, the C-peptide sequence and fold can be subdivided into three distinct parts ('tripartite'). At the functional level, its chaperone-like abilities, self-assembly, and membrane interactions, as well as interactions with relevant proteins can be separately ascribed to these three segments. At the biological level, the assignments are compatible with the suggested roles of C-peptide in granular insulin storage, chaperone-like activities on insulin oligomers, possible depository tendencies, and proposed receptor interactions. Finally, the assignments give interesting parallels to further bioactive peptides, including glucagon and neurotensin. Provided pharmaceutical and clinical trials are successfully completed, the present interpretations should supply mechanistic explanations on C-peptide as a bioactive compound of importance in health and diabetes.
No preview · Article · May 2014 · Biomolecular concepts
[Show abstract][Hide abstract] ABSTRACT: Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated.
Full-text · Article · May 2014 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35-65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic β cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.
Preview · Article · Mar 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
Full-text · Article · Feb 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Chromosomal aneuploidy has been identified as a prognostic factor in the majority of sporadic carcinomas. However, it is not known how chromosomal aneuploidy affects chromosome-specific protein expression in particular, and the cellular proteome equilibrium in general.
The aim was to detect chromosomal aneuploidy-associated expression changes in cell clones carrying trisomies found in colorectal cancer.
We used microcell-mediated chromosomal transfer to generate three artificial trisomic cell clones of the karyotypically stable, diploid, yet mismatch-deficient, colorectal cancer cell line DLD1 - each of them harboring one extra copy of either chromosome 3, 7 or 13. Protein expression differences were assessed by two-dimensional gel electrophoresis and mass spectrometry, compared to whole-genome gene expression data, and evaluated by PANTHER classification system and Ingenuity Pathway Analysis (IPA).
In total, 79 differentially expressed proteins were identified between the trisomic clones and the parental cell line. Up-regulation of PCNA and HMGB1 as well as down-regulation of IDH3A and PSMB3 were revealed as trisomy-associated alterations involved in regulating genome stability.
These results show that trisomies affect the expression of genes and proteins that are not necessarily located on the trisomic chromosome, but reflect a pathway-related alteration of the cellular equilibrium.
No preview · Article · Jan 2014 · Analytical cellular pathology (Amsterdam)
[Show abstract][Hide abstract] ABSTRACT: Abstract Context: Biological material reflecting the in vivo composition of markers provides a high potential for biomarker discovery. Objective: We compared the serum proteome following heat- and nitrogen-preservation, with and without subsequent storage at room temperature. Materials and methods: Serum samples were collected, treated and analysed by two-dimensional gel electrophoresis. Protein spots were identified and confirmed by two mass spectrometry approaches (MALDI & ESI) and subjected to Ingenuity Pathway Analysis. Results: We revealed 24 differentially expressed proteins (p ≤ 0.05) between nitrogen and heat preservation, and 87 between nitrogen and heat preservation with subsequent storage for 120 h at room-temperature. Mass spectrometry identified 25 polypeptides. Pathway analysis resulted in networks maintaining Cellular Assembly and Organization, Movement and Maintenance. Conclusion: Heat-stabilization does not substantially change the short-term proteome composition of serum compared with nitrogen treatment. However, heat-stabilization alone seems insufficient for long-term sample preservation for serum samples. We identified transthyretin and apolipoprotein A-IV as sample quality markers.
No preview · Article · Jul 2013 · Archives of Physiology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Glomerular diseases represent major diagnostic and therapeutic challenges with classification of these diseases largely relying on clinical and histological findings. Elucidation of molecular mechanisms of progressive glomerular disease could facilitate quicker development. High-throughput expression profiling reveals all genes and proteins expressed in tissue and cell samples. These methods are very appropriate for glomerular disease as pure glomeruli can be obtained from kidney biopsies. To date, proteome profiling data are only available for normal glomeruli, but more robust transcriptome methods have been applied to many mouse model and a few human glomerular diseases. Here, we have carried out a meta-analysis of currently available glomerular expression data in normal and diseased glomeruli from mice, rats, and humans using a standardized protocol. The results suggest a potential for glomerular transcriptomics in identifying pathogenic pathways, disease monitoring, and the feasibility to use animal models to study human glomerular disease. We also found that currently there are no specific consensus biomarkers or pathways among different disease data sets, indicating there are likely disease-specific mechanisms and expression profiles. Thus, further transcriptomics and proteomics analysis, especially that of dynamic changes in the diseases, may lead to novel diagnostics tools and specific pharmacologic therapies.Kidney International advance online publication, 19 June 2013; doi:10.1038/ki.2013.169.
No preview · Article · Jun 2013 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: Gradually, the C-peptide part of proinsulin has evolved from being viewed upon as a side product of insulin synthesis and secretion to being considered as a bioactive peptide with endocrine functions. Independent of these, its biophysical properties and peptide interactions point to still further roles of C-peptide, in particular regarding possible links to diabetes-related protein aggregations. Insulin, which can deposit at the injection sites in the treatment of diabetes, and islet amyloid polypeptide (IAPP), which can form amyloid fibrils in the islets of Langerhans in diabetes type 2, are kept nonaggregated by charge-based interactions with C-peptide at defined stoichiometries. It is possible that the conformational stabilization of insulin and IAPP by C-peptide may also counterbalance their aggregational tendencies at the high peptide concentrations in the pancreatic β-cell secretory granules. The concentration imbalances of C-peptide, insulin, and IAPP from the hyperpeptidism early in T2DM patients and the insulin-only injections in T1DM patients may distort equilibria of these peptide interactions and promote protein aggregation. Additionally, the chaperone-like actions of C-peptide may increase bioavailability of insulin supplements given to T1DM patients and prevent the formation of insulin deposits. Similarly, peptide interactions may influence depository tendencies in additional peptide systems. In short, biophysical studies are relevant to establish all roles of peptide imbalances in T1DM and T2DM and associated depository diseases.
No preview · Article · Jun 2013 · Hormone and Metabolic Research
[Show abstract][Hide abstract] ABSTRACT: Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant.
[Show abstract][Hide abstract] ABSTRACT: Different lines of alcohol dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ∼550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins.
No preview · Article · Nov 2012 · Chemico-biological interactions
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Proinsulin processing into insulin and C-peptide in the secretory granules of the pancreatic β-cells occurs under mildly acidic conditions and at high peptide concentrations (> 10 mm). Mature insulin has reduced solubility and a propensity to adopt an amyloid-like structure, but is physiologically released as a mixture of a zinc-containing core and a zinc-free, C-peptide-rich fluid phase. C-peptide is known to function in the insulin secretion, but its exact mode of interaction is not established. We now demonstrate that C-peptide in sub-stoichiometric amount versus insulin coprecipitates with insulin at the pH found in secretory vesicles. Precipitation is reversible and the precipitate is dissolved by elevation of the pH. This effect was found to be dependent on relatively conserved glutamate residues in the otherwise poorly conserved C-peptide. Together, the data show that C-peptide has the ability to influence insulin solubility. The physiological pH changes between insulin processing and release sites may therefore affect the quaternary structure of insulin, as well as the phase transitions during insulin sorting and secretion.
Structured digital abstract:
Insulin and C-peptide bind by molecular sieving (View Interaction: 1, 2) C-peptide and Insulin bind by dynamic light scattering (View Interaction: 1, 2) C-peptide and Insulin bind by fluorescence technology (View Interaction: 1, 2, 3, 4).