Derivation and characterization of a cytocompatible scaffold from human testis.

STUDY QUESTION: Is it possible to derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine? SUMMARY ANSWER: 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.

C-peptide: a molecule balancing insulin states in secretion and diabetes-associated depository conditions.

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 ß-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.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the MDR superfamily.

The MDR superfamily with ~350-residue subunits contains the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase and many more forms. ADH is a dimeric zinc metalloprotein and occurs as five different classes in humans, resulting from gene duplications during vertebrate evolution, the first one traced to ~500 MYA (million years ago) from an ancestral formaldehyde dehydrogenase line. Like many duplications at that time, it correlates with enzymogenesis of new activities, contributing to conditions for emergence of vertebrate land life from osseous fish. The speed of changes correlates with function, as do differential evolutionary patterns in separate segments. Subsequent recognitions now define at least 40 human MDR members in the Uniprot database (corresponding to 25 genes when excluding close homologues), and in all species at least 10888 entries. Overall, variability is large, but like for many dehydrogenases, subdivided into constant and variable forms, corresponding to household and emerging enzyme activities, respectively. This review covers basic facts and describes eight large MDR families and nine smaller families. Combined, they have specific substrates in metabolic pathways, some with wide substrate specificity, and several with little known functions.

Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: implications for an entatic state.

Zinc binding to the peptide replica and analogs to residues 93-115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the epsilon-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site.

Glomerulus proteome analysis with two-dimensional gel electrophoresis and mass spectrometry.

A proteome analysis of mammalian glomeruli was performed in mouse using two-dimensional-gel electrophoresis with separate Coomassie and silver staining and subsequent mass spectrometric identifications. Altogether, 414 protein spots were identified, revealing 232 different proteins representing a wide spectrum of activities, including enzymes (27%), cell-signalling proteins (22%), structural proteins (12%), protein folding and metabolism (13%), cell-growth-related proteins (6%), and replication, transcription and translation (4%). Only 53 of the proteins were detected in another proteome study, showing the value of analyses with different methodologies. However, 50 of the proteins were also identified in a proteome analysis of endothelial cells and 42 in one of glomerular mesangial cells, revealing distinct similarities between these tissues, but also unique differences. Finally, 80 of the proteins were not identified in a separate transcriptome analysis, while 10 of the present proteins were then indentifiable with genes implicated in glomerulus development and function, allowing direct correlation with expression data.

Differential protein expression in pancreatic islets after treatment with an imidazoline compound.

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca(2+) binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1).

Cellular internalization of proinsulin C-peptide.

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37 degrees C for up to 1 h, and was reduced at 4 degrees C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action.

Proinsulin C-peptide and insulin: Limited pattern similarities of interest in inter-peptide interactions but no C-peptide effect on insulin and IGF-1 receptor signaling.

The recently reported influence of proinsulin C-peptide on insulin prompted us to examine structural features of the C-peptide. Four sets of limited pattern similarities towards inter-chain end regions of insulin were noticed, involving secondary structure elements, binding residues and intra- as well as inter-peptide residue similarities. Using surface plasmon resonance, we examined insulin binding to truncated, soluble insulin receptor A and IGF-1 receptor, but C-peptide effects on these bindings were not detectable. Two forms of the insulin receptor, differing in activation of gene transcription with regards to (pre)proinsulin and glucokinase, respectively, were also uninfluenced by C-peptide. We conclude that the pattern similarities, if functional, reflect C-peptide interactions with molecules other than both insulin A and B receptors and IGF-1 receptors. Any such effects are of interest in relation to reported binding interactions between insulin and C-peptide.

Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is secreted in a highly phosphorylated form that binds IGF-I with high affinity and is resistant to proteolysis. We have purified IGFBP-1-specific protease activity from the urine of an individual with multiple myeloma. This protease efficiently cleaves both phosphorylated and non-phosphorylated IGFBP-1 at Ile130-Ser131, generating fragments that together have higher association and dissociation rates for IGFs compared with intact IGFBP-1. The proteolytic fraction contained azurocidin, a protease homologue hitherto considered inactive. After cleavage of IGFBP-1, there was a lower affinity, but higher capacity for IGF-I binding, suggesting both N- and C-terminal fragments may interact with ligand independently. There was decreased inhibition of IGF-II-stimulated cell growth and glucose uptake. Alone, proteolysed IGFBP-1 stimulated glucose uptake in muscle. We conclude that specific cleavage of IGFBP-1 at target tissues is important in cellular growth and metabolism and opens novel strategies for targeting IGFBP-1 in treatment of disease.

Proinsulin C-peptide elicits disaggregation of insulin resulting in enhanced physiological insulin effects.

Using surface plasmon resonance (SPR) and electrospray mass spectrometry (ESI-MS), proinsulin C-peptide was found to influence insulin-insulin interactions. In SPR with chip-bound insulin, C-peptide mixed with analyte insulin increased the binding, while alone C-peptide did not. A control peptide with the same residues in random sequence had little effect. In ESI-MS, C-peptide lowered the presence of insulin hexamer. The data suggest that C-peptide promotes insulin disaggregation. Insulin/insulin oligomer muM dissociation constants were determined. Compatible with these findings, type 1 diabetic patients receiving insulin and C-peptide developed 66% more stimulation of glucose metabolism than when given insulin alone. A role of C-peptide in promoting insulin disaggregation may be important physiologically during exocytosis of pancreatic beta-cell secretory granulae and pharmacologically at insulin injection sites. It is compatible with the normal co-release of C-peptide and insulin and may contribute to the beneficial effect of C-peptide and insulin replacement in type 1 diabetics.

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue.

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.

Vernix caseosa is a white cream-like substance that covers the skin of the foetus and the newborn baby. Recently, we discovered antimicrobial peptides/proteins such as LL-37 in vernix, suggesting host defence functions of vernix. In a proteomic approach, we have continued to characterize proteins in vernix and have identified 20 proteins, plus additional variant forms. The novel proteins identified, considered to be involved in host defence, are cystatin A, UGRP-1, and calgranulin A, B and C. These proteins add protective functions to vernix such as antifungal activity, opsonizing capacity, protease inhibition and parasite inactivation. The composition of the lipids in vernix has also been characterized and among these compounds the free fatty acids were found to exhibit antimicrobial activity. Interestingly, the vernix lipids enhance the antimicrobial activity of LL-37 in vitro, indicating interactions between lipids and antimicrobial peptides in vernix. In conclusion, vernix is a balanced cream of compounds involved in host defence, protecting the foetus and newborn against infection.

Separate functional features of proinsulin C-peptide.

Proinsulin C-peptide influences a number of physiological parameters in addition to its well-established role in the parent proinsulin molecule. It is of interest as a candidate for future co-replacement therapy with insulin for patients with diabetes mellitus type 1, but specific receptors have not been identified and additional correlation with functional effects is desirable. Based on comparisons of 22 mammalian proinsulin variants, we have constructed analogues for activity studies, choosing phosphorylation of mitogen-activated protein kinases (MAPKs) in Swiss 3T3 fibroblasts for functional measurements. In this manner, we find that effective phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at positions 3, 11 and 27 of C-peptide and by the presence of helix-promoting residues in the N-terminal segment. Previous findings have ascribed functional roles to the C-terminal pentapeptide segment, and all results combined therefore now show the importance of different segments, suggesting that C-peptide interactions are complex or multiple.

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells.

AIMS/HYPOTHESIS: Accumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca(2+) influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na(+), K(+)-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells. METHODS: Human renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies. RESULTS: Human C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms delta and epsilon to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC-delta inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-delta. CONCLUSIONS/INTERPRETATION: C-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC-delta and PKC-epsilon, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.

Degradation of proinsulin C-peptide in kidney and placenta extracts by a specific endoprotease activity.

Degradation of proinsulin C-peptide in mouse kidney and human placenta extracts was studied using reverse-phase high-performance liquid chromatography and nano-electrospray mass spectrometry. In total, 15 proteolytic cleavage sites were identified in human and mouse C-peptides. Early sites included the peptide bonds N-terminal of Val/Leu10, Leu12, Leu21, Leu24 and Leu26 in different combinations for the two tissues and two peptides. Notably, these cleavages were N-terminal of a hydrophobic residue, and all but one N-terminal of Leu. A late degradation product of the human peptide detected in the kidney extract was the C-terminal hexapeptide, containing just one residue more than the biologically active C-terminal pentapeptide of C-peptide. We conclude that the degradation of C-peptide in kidney and placenta follows similar patterns, dominated by endopeptidase cleavages N-terminal of Leu.

C-peptide stimulates Na+, K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells.

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na(+), K(+)-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na(+), K(+)-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37 degrees C for 10 min stimulated (86)Rb(+) uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive (86)Rb(+) uptake, C-peptide increased alpha subunit phosphorylation and basolateral membrane (BLM) abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits. The increase in BLM abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits was accompanied by depletion of alpha(1) and beta(1) subunits from the endosomal compartments. C-peptide action on Na(+), K(+)-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na(+), K(+)-ATPase activation, phosphorylation of alpha(1)-subunit and translocation of alpha(1) and beta(1) subunits to the BLM were abolished by a MEK1/2 inhibitor (20 muM PD98059). C-peptide stimulation of (86)Rb(+) uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 muM GF109203X). C-peptide stimulated phosphorylation of human Na(+), K(+)-ATPase alpha subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the alpha subunit of Na(+), K(+)-ATPase.