Jeffrey N. Keen

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP" reductases

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin‐like protein (HMP) by size and N‐terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C‐terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR‐like and haemoglobin‐like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure—function properties of other FNR‐like proteins, including LuxG and VanB, are also discussed.

Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble γ-glutamyl transpeptidase

BackgroundIn Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions.ResultsThirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a γ-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. γ-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG γ-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong γ-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster.ConclusionWe have applied biochemical approaches, not used previously, to characterise prominent D. melanogaster accessory gland products. Of the thirteen accessory gland secreted proteins reported in this study, six were represented in a D. simulans male accessory gland EST library that was biased for male-specific genes. Therefore, the present study has identified seven new secreted accessory gland proteins, including GGT-1, which was not recognised previously as a secreted accessory gland product.

Homology between the pyrazine-binding protein from nasal mucosa and major urinary proteins.

Sequence analysis of the pyrazine‐binding protein from bovine olfactory mucosa reveals marked homology with a family of proteins of unknown function found in the urine of the adult male mouse and rat. In view of the dramatic biological responses to odorants transmitted in male rodent urines, it is proposed that these proteins play important roles in some aspects of odor transmission and reception.

Purification and partial amino acid sequence of human urine protein 1 : Evidence for homology with rabbit uteroglobin

We describe the purification of Urine Protein 1 (UP1), a 14-16 kDa protein which occurs in the urine of patients with renal failure, and therefore may originate from the plasma or kidney. Amino acid sequencing shows that UP1 has significant homology with rabbit uteroglobin, a secretory protein of the uterus (during pregnancy) and lungs (both sexes), and previously identified only in lagomorphs (rabbits, hares, pikas). The finding of a human uteroglobin-like protein, which can be purified from a readily available source, may provide further opportunities to elucidate the, as yet, uncertain physiological functions of uteroglobin.

Complement C3 is a novel plasma clot component with anti-fibrinolytic properties

Background and method: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. Results: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. Conclusion: These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.

Proteomic Screening of a Cell Line Model of Esophageal Carcinogenesis Identifies Cathepsin D and Aldo-Keto Reductase 1C2 and 1B10 Dysregulation in Barrett’s Esophagus and Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barretts esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barretts epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barretts epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.

Peptidyl dipeptidases (Ance and Acer) of Drosophila melanogaster: major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme.

Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro substrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length. Only two of the peptides, [Leu(5)]enkephalinamide and leucokinin-I were cleaved faster by Acer. Increasing NaCl concentration had opposite affects on the cleavage of [Leu(5)]enkephalin and [Leu(5)]enkephalinamide by Acer, decreasing the activity towards [Leu(5)]enkephalin but increasing the activity towards [Leu(5)]enkephalinamide. Of the insect peptides tested, the tachykinin-related peptide, Lom TK-1, proved to be the best substrate for Ance with a k(cat)/K(m) ratio of 0.122s(-1) microM(-1). However, in comparison, the D. melanogaster tachykinins, DTK-1, DTK-2, DTK-3 and DTK-4 were poor Ance substrates. DTK-5 was the best substrate of this family, but the apparent high K(m) for hydrolysis by Ance suggested that this peptide would not be a natural Ance substrate. This low affinity for DTK-5 is the likely reason why the peptide was not rapidly degraded in D. melanogaster hemolymph, where Ance was shown to be a major peptide-degrading activity.

Inactivation of a tachykinin-related peptide: identification of four neuropeptide-degrading enzymes in neuronal membranes of insects from four different orders.

Tachykinin-related peptides (TRP) are widely distributed in the CNS of insects, where they are likely to function as transmitters/modulators. Metabolic inactivation by membrane ecto-peptidases is one mechanism by which peptide signalling is terminated in the CNS. Using locustatachykinin-1 (LomTK-1, GPSGFYGVRamide) as a substrate and several selective peptidase inhibitors, we have compared the types of membrane associated peptidases present in the CNS of four insects, Locusta migratoria, Leucophaea maderae, Drosophila melanogaster and Lacanobia oleracea. A neprilysin (NEP)-like activity cleaving the G-F peptide bond was the major LomTK-1-degrading peptidase detected in locust brain membranes. NEP activity was also found in Leucophaea brain membranes, but the major peptidase was an angiotensin converting enzyme (ACE), cleaving the G-V peptide bond. Drosophila adult head and larval neuronal membranes cleaved the G-F and G-V peptide bonds. Phosphoramidon inhibited both these cleavages, but with markedly different potencies, indicating the presence in the fly brain of two NEP-like enzymes with different substrate and inhibitor specificity. In Drosophila, membrane ACE did not make a significant contribution to the cleavage of the G-V bond. In contrast, ACE was an important membrane peptidase in Lacanobia brain, whereas very little neuronal NEP could be detected. A dipeptidyl peptidase IV (DPP IV) that removed the GP dipeptide from the N-terminus of LomTK-1 was also found in Lacanobia neuronal membranes. This peptidase was a minor contributor to LomTK-1 metabolism by neuronal membranes from all four insect species. In Lacanobia, LomTK-1 was also a substrate for a deamidase that converted LomTK-1 to the free acid form. However, the deamidase was not an integral membrane protein and could be a lysosomal contaminant. It appears that insects from different orders can have different complements of neuropeptide-degrading enzymes. NEP, ACE and the deamidase are likely to be more efficient than the common DPP IV activity at terminating neuropeptide signalling since they cleave close to the C-terminus of the tachykinin, a region essential for maintaining biological activity.

Modification of a gInB‐like gene product by photosynthetic electron transport in the cyanobacterium Synechococcus 6301

Covalent modification of a 13 kDa soluble‐phase protein occurs during adaptation of cells of the cyanobacterium Synechococcus 6301 (mutant AN 112) to light specifically absorbed by photosystem II. This adaptation is accompanied by functional changes indicative of altered excitation energy distribution between the photosystems. The 13 kDa protein is identified by solid‐phase N‐terminal sequencing as a protein related to PII, the glnB gene product of E. coli. In E. coli, the PII protein undergoes uridylylation and acts as a regulator of glutamine synthetase at both the post‐translational and transcriptional levels. The implications of modification of a transcriptional regulator by photosynthetic electron transport are discussed.

Isolation, Cloning, and Characterisation of a trp Homologue from Squid (Loligo forbesi) Photoreceptor Membranes

Abstract: The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92‐kDa component isolated from a detergent‐insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full‐length cDNA, encoding the 92‐kDa component, using both cDNA library screening and 5′‐rapid amplification of cDNA ends (5′‐RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl‐terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl‐terminal domain has been shown to be in the regulation of several ligand‐gated channels. The carboxyl‐terminal domain has been expressed and shown to interact with calmodulin in a calcium‐dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.