Enzyme activity in the small intestine of goat kids during the period of passive immunity acquisition

Enzyme activity of protein and carbohydrate degradation in small intestinal mucosa was investigated in goat kids fed with lyophilized bovine and goat colostrum. At 0,7 and 14 h of life 15 male newborns received 5% of body weight of lyophilized bovine colostrum and 14 goat colostrum, both with 55 mg/mL of IgG. Duodenum, jejunum and ileum samples were collected at 18,36 and 96 h of life. Three animals were sampled at birth, without colostrum intake. Activity of aminopeptidase N and A, dipeptidil peptidase IV, lactase, maltase and sucrase was determined as one international unit per gram of tissue. Intracellular enzymatic activity of acid phosphatase was observed by histochemistry in tissue section. Only the activity of aminopeptidase A in the ileum was affected by treatment, with a greater value for LBC than for GC (P < 0.05). The aminopeptidase N activity was the highest at 36 h in the duodenum (P < 0.05) and lowest at 96 h in the jejunum (P < 0.05). Dipeptidil peptidase IV activity was highest at 36 h in the duodenum (P < 0.05), lowest at 96 h in the jejunum (P < 0.05) and higher at 36 h than at 96 h in the ileum (P < 0.05). Aminopeptidase A activity in the ileum was highest at 36 h (P < 0.05), followed by 18 and 96 h of life (P < 0.05). Lactase activity in the duodenum increased from 18 to 36 h and from 36 to 96 h in the jejunum (P < 0.05). Maltase activity increased only in the duodenum from 18 to 96 h (P < 0.05). Sucrase activity in the jejunum decreased from 18 to 36 h and from 36 to 96 h in the ileum (P < 0.05). At birth, activity of most enzymes was similar to that at later times (P < 0.05). Histochemistry analyses showed a higher frequency of lysosomes with acid phosphatase activity in the duodenum, especially at 36 h of life. In the jejunum, the presence of lysosomes with acid phosphatase activity was the highest at 96 h, followed by 36 and 18 h of life. In the ileum, all samples showed low presence of lysosomes with acid phosphatase activity. These results indicate that lyophilized bovine colostrum, as a heterologous source of antibodies or nutrients, is a possible alternative management tool for goats. The present work also suggests that in the first 4 days of life, enzyme activity in the intestinal epithelium of goats is still not fully stimulated, which is an important characteristic for these animals that depend on macromolecule absorption to acquire passive protection after birth. (C) 2012 Elsevier B.V. All rights reserved.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Peptidomic Analysis of HEK293T Cells: Effect of the Proteasome Inhibitor Epoxomicin on Intracellular Peptides

Peptides derived from cytosolic, mitochondrial, and nuclear proteins have been detected in extracts of animal tissues and cell lines. To test whether the proteasome is involved in their formation, HEK293T cells were treated with epoxomicin (0.2 or 2 mu M) for 1 h and quantitative peptidomics analysis was performed. Altogether, 147 unique peptides were identified by mass spectrometry sequence analysis. Epoxomicin treatment decreased the levels of the majority of intracellular peptides, consistent with inhibition of the proteasome beta-2 and beta-5 subunits. Treatment with the higher concentration of epoxomicin elevated the levels of some peptides. Most of the elevated peptides resulted from cleavages at acidic residues, suggesting that epoxomicin increased the processing of proteins through the beta-1 subunit. Interestingly, some of the peptides that were elevated by the epoxomicin treatment had hydrophobic residues in P1 cleavage sites. Taken together, these findings suggest that, while the proteasome is the major source of intracellular peptides, other peptide-generating mechanisms exist. Because intracellular peptides are likely to perform intracellular functions, studies using proteasome inhibitors need to be interpreted with caution, as it is possible that the effects of these inhibitors are due to a change in the peptide levels rather than inhibition of protein degradation.

Exercise Training Restores Cardiac Protein Quality Control in Heart Failure

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H2O2 release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H2O2 resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca2+-induced permeability transition and reduced H2O2 release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a nonpharmacological tool for heart failure therapy.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D 10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N ¼ 42) or not (small follicle-small CL group; SF-SCL; N ¼ 41) on D 10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 0.33 mm vs. 10.76 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 0.28 pg/mL vs. 1.27 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 0.25 ng/mL vs. 2.62 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

Analysis of the influence of epitope flanking regions on MHC class I restricted antigen presentation

Peptides presented by MHC class I molecules for CTL recognition are derived mainly from cytosolic proteins. For antigen presentation on the cell surface, epitopes require correct processing by cytosolic and ER proteases, efficient TAP transport and MHC class I binding affinity. The efficiency of epitope generation depends not only on the epitope itself, but also on its flanking regions. In this project, the influence of the C-terminal region of the model epitope SIINFEKL (S8L) from chicken ovalbumin (aa 257-264) on antigen processing has been investigated. S8L is a well characterized epitope presented on the murine MHC class I molecule, H-2Kb. The Flp-In 293Kb cell line was transfected with different constructs each enabling the expression of the S8L sequence with different defined C-terminal flanking regions. The constructs differed at the two first C-terminal positions after the S8L epitope, so called P1’ and P2’. At these sites, all 20 amino acids were exchanged consecutively and tested for their influence on H-2Kb/S8L presentation on the cell surface of the Flp-In 293Kb cells. The detection of this complex was performed by immunostaining and flow cytometry. The prevailing assumption is that proteasomal cleavages are exclusively responsible for the generation of the final C-termini of CTL epitopes. Nevertheless, recent publications showed that TPPII (tripeptidyl peptidase II) is required for the generation of the correct C-terminus of the HLA-A3-restricted HIV epitope Nef(73-82). With this background, the dependence of the S8L generation on proteasomal cleavage of the designed constructs was characterized using proteasomal inhibitors. The results obtained indicate that it is crucial for proteasomal cleavage, which amino acid is flanking the C-terminus of an epitope. Furthermore, partially proteasome independent S8L generation from specific S8L-precursor peptides was observed. Hence, the possibility of other existing endo- or carboxy-peptidases in the cytosol that could be involved in the correct trimming of the C-terminus of antigenic peptides for MHC class I presentation was investigated, performing specific knockdowns and using inhibitors against the target peptidases. In parallel, a purification strategy to identify the novel peptidase was established. The purified peaks showing an endopeptidase activity were further analyzed by mass spectrometry and some potential peptidases (like e.g. Lon) were identified, which have to be further characterized.

Funktionelle Analyse der Meprin-Metalloproteasen alpha und beta hinsichtlich ihrer physiologischen Regulation und biologischen Bedeutung

Im Rahmen dieser Arbeit ist es gelungen, ein besseres Verständnis der beiden Metalloproteasen Meprin α und β in ihrem proteolytischen Netzwerk hinsichtlich ihrer physiologischen Regulation durch endogene Inhibitoren, wie auch der biologischen Funktion von Meprin α für den Prozess der Angiogenese, zu erlangen. rnMit der Analyse des ersten identifizierten endogenen Meprin-Inhibitors Fetuin-A gelang die Bestimmung der Ki-Werte für Meprin α mit 4,2 x 10-5 M und 1,1 x 10-6 M für Meprin β. Des Weiteren konnte für Meprin β eine Schnittstelle im Fetuin-A validiert werden. Mit der Identifizierung von Cystatin C, einem Cystein-Protease-Inhibitor als endogener Inhibitor der Metalloprotease Meprin α, mit einem Ki-Wert von 8,5 x 10-6 M, wurden erstmals Proteasefamilie-übergreifende Inhibitionsmechanismen für Metalloproteasen offenbart.rnDie Analyse von drei potentiellen Meprin-Inhibitoren, identifiziert als Substrate in einem neuen Proteomics-Analyse-Verfahren terminal amine isotopic labeling of substrates (TAILS), ermöglichte die Charakterisierung von Elafin als spezifischen Meprin α-Inhibitor. Für Elafin ist es außerdem gelungen, die durch TAILS ermittelte Schnittstelle für Meprin α mittels Edman Sequenzierung zu validieren. Der secretory leukocyte peptidase inhibitor (SLPI), ein Elafin-Homolog, konnte als weiteres Meprin α-Substrat bestätigt werden. Außerdem gelang es, die Meprin α-Schnittstelle im SLPI zu validieren.rnEin weiteres Ziel dieser Arbeit war, ein besseres Verständnis der biologischen Funktion der Metalloprotease Meprin α zu erlangen. Hier konnte in vivo eine stark pro-angiogene Wirkung von Meprin α gezeigt werden und erstmals die Expression von Meprin α, jedoch nicht von Meprin β, in Endothelzellen nachgewiesen werden. Zugleich konnte mit der Analyse der durch die TAILS-Methode identifizierten pro-angiogenen Substrate vascular endothelial growth factor A (VEGF-A) und connective tissue growth factor (CTGF) der Regulationsmechanismus von Meprin α in der Angiogenese identifiziert werden. So ist Meprin α durch die Spaltung von CTGF in der Lage VEGF-A – gebunden und inhibiert im Komplex mit CTGF – durch proteolytische Spaltung von CTGF wieder freizusetzen. Somit wird die inhibierte VEGF-A-Aktivität wieder vollständig hergestellt. rnMit der Charakterisierung der ersten endogenen Meprin-Inhibitoren ist es gelungen, zu einem besseren Verständnis der endogenen Regulation der Meprine beizutragen und eine Proteasefamilie-übergreifende endogene Regulation aufzuzeigen. Mit der Entdeckung von Meprin α als pro-angiogene Protease und der Entschlüsselung des angiogenen Regulationsmechanismus konnte eine essentielle biologische Bedeutung dieser Protease beschrieben werden.rn

Isolierung und Charakterisierung der ER-residenten Aminopeptidase ERMP1 und Untersuchung ihrer Funktion in der Prozessierung MHC I restringierter CTL Epitope

Die effiziente Generierung von Peptid-Epitopen aus zelleigenen oder viralen Proteinen für die Präsentation auf „Major Histocompatibility Complex I“ (MHC I) Molekülen ist essentiell für die Aktivierung des adaptiven Immunsystems und die Effektorfunktion der CD8+ zytotoxischen T-Zellen (CTLs). CTLs erkennen diese Peptide in Kontext mit MHC I Molekülen über ihren spezifischen T-Zellrezeptor (TCR). Die Generierung dieser Epitope ist das Resultat eines komplexen proteolytischen Prozesses, der im Zytosol und im endoplasmatischen Retikulum (ER) stattfindet. Im Zytosol generiert das Proteasom N-terminal verlängerte Epitop-Vorläufer. Diese werden durch weitere zytosolische Proteasen abgebaut, es sei denn, sie werden durch den „transporter associated with antigen processing“ (TAP) in das ER transportiert. Dort werden sie durch Aminopeptidasen getrimmt, um den Bindungsvoraussetzungen der MHC I Moleküle zu genügen. Im murinen System ist die „ER aminopeptidase associated with antigen processing“ (ERAAP) die bislang einzige beschriebene Aminopeptidase, die dieses N-terminale Trimming von CTL Epitopen vermitteln kann. Das Profil der proteolytischen Aktivität in angereichertem murinen ER kann jedoch nicht allein durch die Aktivität von ERAAP erklärt werden, was auf die Anwesenheit weiterer Aminopeptidasen mit einer potentiellen Funktion in der Antigenprozessierung hinweist. In dieser Arbeit konnte die immunologisch bislang noch nicht beschriebene Aminopeptidase ERMP1 (endoplasmic reticulum metallopeptidase 1) im murinen ER identifiziert werden. Nach Aufreinigung muriner Mikrosomen und anschließender Anionenaustausch-Chromatographie wurden die gesammelten Fraktionen mit fluorogenen Substraten auf Aminopeptidase-Aktivität getestet. Durch massenspektrometrische Analyse konnten in den beobachteten Peaks die schon beschriebenen Aminopeptidasen ERAAP, die „insulin regulated aminopeptidase“ IRAP und die immunologisch bislang nicht beschriebene Aminopeptidase ERMP1 identifiziert werden. Durch Fluoreszenzmikroskopie konnte die intrazelluläre Lokalisation von ERMP1 im ER durch Kolokalisation mit TAP verifiziert werden. Wie viele Komponenten des MHC I Prozessierungsweges wird auch die Expression von ERMP1 durch IFN-γ stimuliert. Dies macht ERMP1 zu einer potentiellen zweiten trimmenden Aminopeptidase im murinen ER. Überexpression von ERMP1 hat einen allelspezifischen Einfluss auf die globale MHC I Präsentation auf der Zelloberfläche und durch Überexpression und shRNA vermitteltes gene silencing konnte außerdem ein epitopspezifischer Effekt nachgewiesen werden. Da N-terminales Trimming durch ERAAP mit der Evasion von Tumoren und veränderter Immundominanz assoziiert wird, ist die detaillierte Charakterisierung der Aminopeptidase ERMP1 ein wichtiger Schritt zum Verständnis der MHC I Antigen-Prozessierung und der Generierung von CTL Epitopen im ER.

Leishmania major parasites and their interaction with human macrophages

First both life stages of Leishmania major (L. major) FEBNI parasites, promastigotes as well as amastigotes, were characterized. We found that the virulence marker GP63 and cysteine peptidase b (Cpb) were higher expressed by axenic amastigotes as compared to promastigotes. In addition to the L. major FEBNI strain, we applied and successfully modified our novel in vitro method to generate axenic amastigotes of the L. major Friedlin and 5ASKH strains. Interestingly, these L. major strains needed another temperature to be transferred into amastigotes in the axenic culture system. Investigating apoptosis mechanisms in both parasite life stages of L. major FEBNI we found both ROS dependent and independent cell death mechanisms. Focusing on promastigote and amastigote interaction with pro-inflammatory (MF I) and anti-inflammatory (MF II) macrophages we found amastigotes to be more infective as compared to promastigotes. Moreover, we could demonstrate that pro-inflammatory MF I were less susceptible to infection than anti-inflammatory MF II. Finally we investigated parasite stage-specific responses of MF I + II and their defense mechanisms against L. major. Using knockdown techniques for primary human macrophages we identified a new mechanism enabling intracellular killing of promastigotes inside MF I. This mechanism depends on the antimicrobial molecule cathelicidin (LL-37).

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

The role of self-monitoring of blood glucose in glucagon-like Peptide-1-based treatment approaches: a European expert recommendation

The role of glucagon-like peptide (GLP)-1-based treatment approaches for type 2 diabetes mellitus (T2DM) is increasing. Although self-monitoring of blood glucose (SMBG) has been performed in numerous studies on GLP-1 analogs and dipeptidyl peptidase-4 inhibitors, the potential role of SMBG in GLP-1-based treatment strategies has not been elaborated. The expert recommendation suggests individualized SMBG strategies in GLP-1-based treatment approaches and suggests simple and clinically applicable SMBG schemes. Potential benefits of SMBG in GLP-1-based treatment approaches are early assessment of treatment success or failure, timely modification of treatment, detection of hypoglycemic episodes, assessment of glucose excursions, and support of diabetes management and diabetes education. Its length and frequency should depend on the clinical setting and the quality of metabolic control. It is considered to play an important role for the optimization of diabetes management in T2DM patients treated with GLP-1-based approaches.

Proteolytic processing of chemokines: implications in physiological and pathological conditions

Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and fine-tunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing.

Projection structure of DtpD (YbgH), a prokaryotic member of the peptide transporter family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as beta-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 A resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.