Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.     

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.     

Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation

The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC(50) about of 5nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.     

Computational model of EGFR and IGF1R pathways in lung cancer: a Systems Biology approach for Translational Oncology

In this paper we propose a Systems Biology approach to understand the molecular biology of the Epidermal Growth Factor Receptor (EGFR, also known as ErbB1/HER1) and type 1 Insulin-like Growth Factor (IGF1R) pathways in non-small cell lung cancer (NSCLC). This approach, combined with Translational Oncology methodologies, is used to address the experimental evidence of a close relationship among EGFR and IGF1R protein expression, by immunohistochemistry (IHC) and gene amplification, by in situ hybridization (FISH) and the corresponding ability to develop a more aggressive behavior. We develop a detailed in silico model, based on ordinary differential equations, of the pathways and study the dynamic implications of receptor alterations on the time behavior of the MAPK cascade down to ERK, which in turn governs proliferation and cell migration. In addition, an extensive sensitivity analysis of the proposed model is carried out and a simplified model is proposed which allows us to infer a similar relationship among EGFR and IGF1R activities and disease outcome.     

Sub-cellular Localization of Calcium in Azolla-Anabaena Symbiosis by Chlortetracycline,ESI and EELS

The subcellular localization of calcium in cells of symbiotic partners located within leaf cavities of Azolla was investigated by using chlorotetracycline, ESI and EELS analysis. Loosely membrane-bound calcium was evidenced by using CTC or EGTA and CTC, in cytoplasmic regions of Azolla hair cells and in cytoplasm of the cyanobiont. Tightly membrane-bound calcium revealed by CTC, and ESI and EELS analysis, was observed in cyanophycin granules and carboxysomes of the cyanobiont. A third calcium type, revealed by ESI and EELS analysis, was localized at the level of cell walls of simple and branched Azolla hairs, in the envelope of heterocysts, and in the cell walls of the cyanobiont.     

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3′UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.Key words: melanoma, microRNA profiling, biomarker, acral, KRAS-variant, SNP     

Oxidative stress in cardiovascular disease: myth or fact?

Oxidative stress is a mechanism with a central role in the pathogenesis of atherosclerosis, cancer, and other chronic diseases. It also plays a major role in the aging process. Ischemic heart disease is perhaps the human condition in which the role of oxidative stress has been investigated in more detail: reactive oxygen species and consequent expression of oxidative damage have been demonstrated during post-ischemic reperfusion in humans and the protective role of antioxidants has been validated in several experimental studies addressing the pathophysiology of acute ischemia. Although an impressive bulk of experimental studies substantiate the role of oxidative stress in the progression of the damage induced by acute ischemia, not a single pathophysiologic achievement has had a significant impact on the treatment of patients and randomized, controlled clinical trials, both in primary and secondary prevention, have failed to prove the efficacy of antioxidants in the treatment of ischemic cardiovascular disease. This dichotomy, between the experimental data and the lack of impact in the clinical setting, needs to be deeply investigated: certainly, the pathophysiologic grounds of oxidative stress do maintain their validity but the concepts of the determinants of oxidative damage should be critically revised. In this regard, the role of intermediate metabolism during myocardial ischemia together with the cellular redox state might represent a promising interpretative key.     

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.     

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis.A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation.These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.     

Mn(II) Oxidation Is Catalyzed by Heme Peroxidases in “Aurantimonas manganoxydans” Strain SI85-9A1 and Erythrobacter sp. Strain SD-21

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “Aurantimonas manganoxydans” strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca²⁺ ions and H₂O₂ to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min⁻¹ mg⁻¹. Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.Mn(III,IV) oxides (Mn oxides) and soluble Mn(III) complexes are the strongest oxidizing agents in the environment after oxygen and play an important role in many biogeochemical cycles (25). At pH 7, they can oxidize metals, catalyze the formation of humic substances and organic nitrogen complexes, and oxidatively degrade humic and fulvic acids to bioavailable low-molecular-weight organic compounds (6, 38, 40). Geochemical cycling of Mn oxides can also control the distribution of many trace elements, as Mn minerals are highly charged and can adsorb and concentrate metals (22). In the pH range of aerobic natural waters (pH 6 to 8), chemical oxidation of Mn(II) is slow, but in the presence of Mn(II)-oxidizing microorganisms, the rate can be 4 to 5 orders of magnitude higher (30, 39, 48).Multicopper oxidases (MCOs) are the only identified proteins from bacteria capable of manganese oxidation. These enzymes are a class of proteins that utilize copper as a cofactor to perform four one-electron substrate oxidations, thereby reducing molecular oxygen to H₂O (37). Generally, MCOs oxidize organic compounds such as phenolics, but some fungal MCOs (laccases) that can oxidize Mn(II) to Mn(III) and Mn(IV) have been described previously (21, 36, 28). Bacterial MCOs involved with Mn oxidation have been genetically identified in Pseudomonas putida strains MnB1 and GB1 (cumA), Leptothrix discophora SS-1 (mofA), Bacillus sp. strain SG-1 (mnxG), and the alphaproteobacterium Pedomicrobium sp. strain ACM 3067 (moxA) (35). None of these MCOs share significant homology except for their copper binding motifs (35), and only in Bacillus sp. (11) have MCOs been directly linked to Mn oxidation.Another class of proteins known in eukaryotes to oxidize manganese, but not commonly identified to be involved in bacterial Mn oxidation, are heme-containing manganese peroxidases (MnPs) (5, 33). These enzymes are extremely important for the degradation activities of lignin-degrading fungi. The MnP from the basidiomycete Phanerochaete chrysosporium has a single Mn(II) binding site near the heme and produces two Mn(III) equivalents at the expense of one H₂O₂ equivalent (18, 34, 45). MnPs and MCOs are able to work in concert, with the MnP utilizing H₂O₂ produced by the MCO-catalyzed Mn(II) oxidation (36). Both types of protein produce Mn(III). While MnPs are best known to occur in fungi, a similar mechanism has been reported for a catalase/peroxidase from Mycobacterium (27), and a catalase-peroxide mechanism was suggested to be involved in Fe and Mn oxidation in Arthrobacter (13).A search for MCO-like genes in the draft genome sequence of the Mn(II)-oxidizing marine alphaproteobacterium “Aurantimonas manganoxydans” strain SI85-9A1 revealed duplicate copies of moxA-like genes (12). Initial attempts to isolate the Mn oxidase enzyme focused on the secreted proteins. Two regions were identified by a native polyacrylamide gel electrophoresis (PAGE) in-gel activity assay to have active Mn(II) oxidation, one at >250 kDa and the other at approximately 50 kDa. The active areas were excised from the gel, digested with trypsin, and analyzed with tandem mass spectrometry (MS/MS) but no peptides could be identified (12). In the same study, five bands from a corresponding Coomassie-stained sodium dodecyl sulfate (SDS)-PAGE gel returned identifications of peptides from a putative Ca²⁺ binding heme peroxidase, but no attempt was made to connect this protein to the active bands from the in-gel activity assay because they would have migrated differently (Coomassie bands were denatured protein, the in-gel activity assay native proteins).Based on the genome data, the expected size of the MoxA-like proteins was approximately 50 kDa, similar to the size of MoxA from Pedomicrobium sp. strain ACM 3067 and similar in size to the most active region from the in-gel activity assay. It was then inferred that the Mn oxidase from A. manganoxydans strain SI85-9A1 could be a Mox ortholog with an estimated size of 50 kDa that may be part of a larger >250-kDa holoenzyme (12). The experimentally identified Ca²⁺ binding heme peroxidase was suggested to be involved with the biodegradation of complex organics utilizing H₂O₂ abiotically generated by the Mn(III) produced by the Mn(II)-oxidizing MoxA-like protein (after the mechanism described by Schlosser and Höfer [36]) (12).In Erythrobacter sp. strain SD-21, the Mn oxidase enzyme was found in both the soluble and excreted fractions, suggesting that the activity may be loosely associated with the cell surface (23). Protein chromatography was employed to identify the Mn oxidase in this organism but did not conclusively implicate an MCO. The enzyme was partially purified and was found to be associated with a quinone cofactor, PQQ, that stimulated manganese oxidation in partially pure protein extracts and rescued the manganese oxidation activity in a mutant strain of Pseudomonas putida MnB1. Mn oxidation was not stimulated in vitro when copper was added, and activity was vastly decreased in the presence of MCO and quinone inhibitors. The addition of o-phenanthroline, a copper chelator and potent inhibitor of Mn oxidation in P. putida GB-1 (32), inhibited Mn oxidation only partially at concentrations far in excess of those required for GB-1. The absorbance spectrum of the partially purified protein extract did not show characteristics of an MCO, and the activity of the cell extract was between 7- and 30-fold higher than the activity measured for Mn-oxidizing organisms containing MCOs. Although the evidence pointed away from MCO involvement, H₂O₂ did not stimulate activity as expected if the enzyme was an MnP (23).A. manganoxydans strain SI85-9A1 is not easily amenable to genetic techniques, and thus, isolation of the manganese oxidase was performed through protein chromatography techniques. Since early studies failed to conclusively identify the Mn oxidase, it was decided to fractionate the proteins in the organism to localize the activity and purify the protein from the active fraction. In this work, we report the significant purification of the Mn-oxidizing protein leading to its identification as an animal heme peroxidase with multiple calcium binding motifs, and localization of the protein to the outer membrane as a peripheral membrane protein. We revisit the Mn oxidase from Erythrobacter sp. strain SD-21 (also genetically recalcitrant) and identify the protein from an active Mn-oxidizing band with a native PAGE in-gel activity assay. This protein is also an animal heme peroxidase with calcium binding motifs.