Differential Distribution and Activity Profile of Acylpeptide Hydrolase in the Rat Seminiferous Epithelium.

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag).

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.

Probing the role of an invariant active site His in family GH1 ß-glycosidases.

The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing ß-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recognised as substrate. Most GH1 ß-glycosidases bind their substrates with similar specificity through invariant amino acid residues. Despite extensive studies, the clear identification of the roles played by each of these residues in the recognition of different glycones is not always possible. We demonstrated here that a histidine residue, completely conserved in the active site of the enzymes of this family, interacts with the C2-OH of the substrate in addition to the C3-OH as previously shown by 3 D-structure determination.

The Sulfolobus solfataricus RecQ-like DNA helicase Hel112 inhibits the NurA/HerA complex exonuclease activity.

ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3' single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Low Erythrocyte Levels of Proteasome and Acyl-Peptide Hydrolase (APEH) Activities in Alzheimer's Disease: A Sign of Defective Proteostasis?

Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia. To date, there are no definitive diagnostic tests that can predict or assess onset and progression of the disease. Blood biomarkers for AD are being sought for many years but their identification remains a challenging task. In this study, we investigated the potential relationship between AD and levels of acyl-peptide hydrolase (APEH) and proteasome in erythrocyte samples of 52 participants (26 AD and 26 cognitively healthy controls). A statistically significant decrease in proteasome and exopeptidase/endopeptidase APEH activities was found in AD samples compared to those of healthy controls. Moreover, in contrast to what was observed for proteasome transcripts, APEH activities reduction in AD patients was unrelated to its gene expression levels, suggesting the occurrence of posttranslational modifications or the expression of endogenous inhibitors that might impair enzyme activity. These preliminary data further support a relationship between the APEH-proteasome system and AD molecular players, providing the first evidence of its potential use as a novel blood-based indicator for the routine detection of AD.

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli.

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, ß-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s-1 and kcat/Km = 1.1 × 108 M-1 s-1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas).

The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO2 hydration reaction, based on the measured kinetic parameters, i.e., kcat = 1.0 × 106 s-1 and kcat/KM = 1.2 × 108 M-1·s-1. CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76-87 µM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes.

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s-1 and kcat/Km of 3.5 × 107 M-1 ×s-1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s-1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s-1 and kcat/Km of 3.6 × 107 M-1 × s-1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

Introducing transgalactosylation activity into a family 42 ß-galactosidase.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a ß-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase.

BACKGROUND: Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. METHODS: As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. RESULTS: Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. CONCLUSIONS: Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. GENERAL SIGNIFICANCE: A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii.

Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its 'uncommon' ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery.

A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering.

In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-tag™ protein, but showing high intrinsic stability typical of proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as protein-tag for organisms living in extreme environments.