Proteolytic Products Research Articles

The Fate of Nascent APP in Hippocampal Neurons: A Live Cell Imaging Study.

Amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD) because its proteolytic products form amyloid plaques and its mutations are linked to familial AD patients. As a membrane protein, APP is involved in neuronal development and plasticity. However, it remains unclear how nascent APP is distributed and transported to designated membrane compartments to execute its diverse functions. Here, we employed a dual-tagged APP fusion protein in combination with a synaptic vesicle marker to study the surface trafficking and cleavage of APP in hippocampal neurons immediately after its synthesis. Using long-term time-lapse imaging, we found that a considerable amount of nascent APP was directly transported to the somatodendritic surface, from which it propagates to distal neurites. Some APP in the plasma membrane was endocytosed and some was cleaved by α-secretase. Hence, we conclude that surface transportation of APP is a major step preceding its proteolytic processing and neuritic distribution.

Two New Secreted Proteases Generate a Casein-Derived Antimicrobial Peptide in Bacillus cereus Food Born Isolate Leading to Bacterial Competition in Milk.

Milk and dairy products harbor a wide variety of bacterial species that compete for both limited resources and space. Under these competitive conditions, bacteria develop specialized mechanisms to protect themselves during niche colonization and nutrient acquisition processes. The bacterial antagonism mechanisms include the production of antimicrobial agents or molecules that facilitate competitor dispersal. In the present work, a bacterial strain designated RC6 was isolated from Ricotta and identified as Bacillus cereus. It generates antimicrobial peptide (AMP) when grown in the presence of casein. The AMP was active against several species of Bacillus and Listeria monocytogenes. MALDI-TOF analysis of the RP-HPLC purified fractions and amino acid sequencing revealed a molecular mass of 751 Da comprised of a 6-residue sequence, YPVEPF. BLAST analysis showed that the AMP corresponds to the fractions 114–119 of bovine β-casein and represents the product of a specific proteolysis. Analysis of the purified proteolytic fractions from the B. cereus RC6 culture supernatant indicated that the presence of at least two different endoproteases is crucial for the generation of the AMP. Indeed, we were able to identify two new candidate endoproteases by means of genome sequencing and functional assignment using a 3D structural model and molecular docking of misannotated hypothetical proteins. In this light, the capacity of B. cereus RC6 to generate antimicrobial peptides from casein, through the production of extracellular enzymes, presents a new model of antagonistic competition leading to niche colonization. Hence, as a dairy product contaminant, this strategy may enable proteolytic B. cereus RC6 niche specialization in milk matrices.

Abstract 547: High-density Lipoprotein Particle Diversity From Selective Combinatorial Assembly of Particle Constituents Result in a Dynamic Particle Population

Introduction: Mass spectrometry is starting to elucidate the extent of lipoprotein diversity. It is believed that HDL is comprised of proteins encoded by >100 genes and ~200 distinct lipid species in an undetermined number of combinations. Multiple proteoforms derived from a single gene contribute to particle complexity, confound proteome identification and makes differentiation of distinct subpopulations a challenge. Subtle proteoform variants can account for alterations in particle physicochemical properties and critical functions. Refining high-definition constituent identification within a structured atlas may reveal mechanism-based pathophysiology of a variety of diseases. Hypothesis: The ongoing identification of HDL constituents necessitates an effort to catalogue, categorize, map and relate entities in a framework that organizes this knowledge in a form of testable observations, explanations and predictions. Defining constituent interactions will advance knowledge and guide inquiry that enables HDL research, clinical diagnostics and subsequent therapeutic strategies. Methods: Informatic analysis of the literature produced an HDL proteome reference set. Proteome specific analysis of gene isoforms, proteolytic products, amino acid modifications and cSNPs were used to prepare a proteoform index. Theoretical tryptic peptide maps were generated and referenced to the PeptideAtlas and available published mass spec peptide lists. Results: Over 380 non-immunoglobulin proteins were identified in high-density lipoprotein fractions from human samples. A consensus-based scoring produced an “unofficial” list of 122 genes. UniProtKB was used to expand an index of potential proteoforms and derive a theoretical peptide mass library used to map empirically defined peptide spectral lists. Conclusions: Utilizing a reductionism model to HDL measurement has proven insufficient and undermines the application of Precision Medicine for CAD. Understanding the health benefits of HDL requires clinical diagnostics that captures particle diversity and population heterogeneity. Constructing a conceptual framework that unifies a constituent map is the initial step to resolving their relational context and functional consequences.

Abstract 039: A Small Fragment Derived from von Willebrand Factor Improves Survival in a Mouse Model of Endotoxemia-Induced Disseminated Intravascular Coagulation

Introduction: Disseminated intravascular coagulation (DIC) is a life-threatening condition that results in wide-spread clot formation and organ failure. Sepsis-induced DIC is associated with a high mortality rate, prompting the need for an effective therapeutic intervention. Our lab has identified the A2 domain of the von Willebrand factor (VWF) protein as a potent treatment for sepsis-induced DIC. Historically, VWF has only been described as a contributor of clot formation; however, our lab has demonstrated that purified recombinant full length A2 domain of the VWF protein can dampen fibrin-rich microthrombi formation in mice with endotoxin. The mode of action appears to be via the interaction with the cell-surface expressed vimentin on endothelium, inhibiting VWF strings formation and thus, platelet adhesion. Goal: Since the VWF protein is naturally cleaved in circulation by the enzyme ADAMTS-13 via the A2 domain, one can argue whether the beneficial effect observed with full len gth A2 domain is terminated by the cleavage of ADAMTS-13. We produced recombinant proteins encompassing the sequences of the two proteolytic products of the A2 domain; the A2Nt and A2Ct proteins. I will identify which fragment retains the mode of action of the full length A2 domain in a mouse model of endotoxin-induced DIC. Methods: The recombinant A2 and vimentin proteins were produced using the bacterial expression system. Protein: protein interactions were determined by optical interferometry. The endotoxin-induced DIC model was achieved by injecting C57B/6 mice with lipopolysaccharide (LPS). Treatment with A2 or its fragments was started approximately 2 hours after LPS insult. Results: The A2N t protein has a higher affinity for vimentin than the A2Ct. Treatment of A2Nt in a LPS-induced DIC mouse model was effective in maintaining survival in males, a comparable finding that mimics treatment of mice with full-length recombinant A2. Interestingly, A2Nt showed less survival than full length A2 in females. These findings highlight the potential differences in therapy responses between genders, an occurrence that some studies have described to also occur in humans.

Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix

Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis – a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12′s proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12′s proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12′s proangiogenic effect is mediated through several molecular mechanisms.

Prion-like propagation of \u03b2-amyloid aggregates in the absence of APP overexpression

The amyloid cascade hypothesis posits that the initiating event in Alzheimer’s disease (AD) is the aggregation and deposition of the β-amyloid (Aβ) peptide, which is a proteolytic cleavage product of the amyloid precursor protein (APP). Mounting evidence suggests that the formation and spread of prion-like Aβ aggregates during AD may contribute to disease progression. Inoculation of transgenic mice that overexpress APP with pre-formed Aβ aggregates results in the prion-like induction of cerebral Aβ deposition. To determine whether Aβ deposition can also be induced when physiological APP levels are present in the brain, we inoculated AppNL-F mice, a knock-in model of AD that avoids potential artifacts associated with APP overexpression, with Aβ aggregates derived from the brains of AD patients or transgenic mice. In all cases, induced Aβ deposition was apparent in the corpus callosum, olfactory bulb, and meningeal blood vessels of inoculated mice at 130–150 days post-inoculation, whereas uninoculated and buffer-inoculated animals exhibited minimal or no Aβ deposits at these ages. Interestingly, despite being predominantly composed of protease-resistant Aβ42 aggregates, the induced parenchymal Aβ deposits were largely diffuse and were unreactive to an amyloid-binding dye. These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Aβ deposition. Accordingly, spreading of Aβ deposition may contribute to disease progression in AD patients.

Coadministration of Intranasally Delivered Insulin and Proinsulin C-Peptide to Rats with Types 1 and 2 Diabetes Mellitus Restores Their Metabolic Parameters

The C-peptide, the product of proinsulin proteolysis, not only is a signal molecule, but also, forming a complex with insulin, is able to modulate the signaling functions of insulin. The signaling systems sensitive to insulin in the hypothalamus and other brain areas are among the targets of insulin. We hypothesized that in systemic deficiency of insulin and C-peptide in the type 1 diabetes mellitus (DM) and in severe forms of the type 2 DM, the increase in the level of C-peptide in the CNS will improve central effects of insulin, including its influence on peripheral metabolism. To verify this, the influence of separate and co-administration of intranasal insulin (II) and C-peptide (IP) on their metabolic parameters and sensitivity to insulin in rats with acute and mild type 1 DM induced by the treatment with streptozotocin at the doses of 60 and 35 mg/kg and in rats with neonatal type 2 DM corresponding to severe long-term form of type 2 DM in human was studied. The treatment of animals with II and IP was carried out for 7 days in the daily doses of 20 and 10 μg/rat, respectively. The co-administration of II and IP leading to an increase of insulin and C-peptide levels in the brain was most effective. In rats with type 1 DM treated with the combination of II plus IP, hyperglycemia was decreased and weight loss was prevented. In rats with type 2 DM, co-administration of II and IP led to the normalization of glucose homeostasis and the increase in insulin sensitivity, as shown by glucose-tolerance and insulin-glucose tolerance tests, and to improvement of lipid metabolism, as demonstrated by the decrease in the atherogenic index. The effectiveness of monotherapy with II was lower than in the case of a combination of II+IP, while monotherapy with C-peptide had little effect on the indicators studied. Thus, the simultaneous increase of insulin and C-peptide levels in the brain in the conditions of their deficiency in diabetic pathology can be considered as one of the promising approaches to restore the central insulin-dependent regulation of peripheral metabolism and to improve the utilization of glucose in different forms of DM.

Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated.

Catalpol Inhibits Amyloid-β Generation Through Promoting α-Cleavage of APP in Swedish Mutant APP Overexpressed N2a Cells.

Amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer’s disease (AD), due to its neurotoxicity. Thus, blocking Aβ generation and aggregation in the brain has been realized as an efficient way for the prevention of AD. The natural product catalpol, isolated from Rehmannia glutinosa, has shown neuroprotective activities through inhibiting soluble Aβ production, degrading Aβ peptide, and attenuating Aβ toxicity and neuroinflammatory responses. In the present study, we aimed to evaluate whether catalpol reduce Aβ generation associated with regulating amyloid precursor protein (APP) proteolytic processing. By using Swedish mutant APP overexpressed N2a (SweAPP N2a) cells treated with catalpol, we found that catalpol was not able to reduce the expression levels of β-secretase (BACE-1) and γ-secretase (PS1, APH-1, PEN-2 and Nicastrin). By contrast, catalpol had a significant promotion effect on the expression of α-secretase (ADAM10) and its proteolytic products, sAPPα and C83, suggesting that catalpol reduced the production of Aβ might be involved in non-amyloidogenic APP pathway. In addition, we confirmed that the extracellular signal-related kinase/cAMP-response element binding protein (ERK/CREB) signaling pathways were responsible for the up-regulation of ADAM10 in catalpol-treated SweAPP N2a cells. The present data, for the first time, have demonstrated that the effect of catalpol on the inhibiting Aβ generation might be closely related to α-cleavage of APP processing.

Molecular partners of hNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, suggest its involvement in distinct cellular processes relevant to congenital disorders of glycosylation, cancer, neurodegeneration and a variety of further pathologies.

This study provides first insights into the involvement of hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID and yeast ALG3 gene, in various putative molecular networks. HNOT/ALG3 encodes two translated transcripts encoding precursor proteins differing in their N-terminus and showing 33% identity with the yeast asparagine-linked glycosylation 3 (ALG3) protein. Experimental evidence for the functional homology of the proteins of fly and man in the N-glycosylation has still to be provided. In this study, using the yeast two-hybrid technique we identify 17 molecular partners of hNOT-1/ALG3-1. We disclose the building of hNOT/ALG3 homodimers and provide experimental evidence for its in vivo interaction with the functionally linked proteins OSBP, OSBPL9 and LRP1, the SYPL1 protein and the transcription factor CREB3. Regarding the latter, we show that the 55 kDa N-glycosylated hNOT-1/ALG3-1 molecule binds the N-glycosylated CREB3 precursor but does not interact with CREB3's proteolytic products specific to the endoplasmic reticulum and to the nucleus. The interaction between the two partners is a prerequisite for the proteolytic activation of CREB3. In case of the further binding partners, our data suggest that hNOT-1/ALG3-1 interacts with both OSBPs and with their direct targets LRP1 and VAMP/VAP-A. Moreover, our results show that various partners of hNOT-1/ALG3-1 interact with its diverse post translationally processed products destined to distinct cellular compartments. Generally, our data suggest the involvement of hNOT-1/ALG3-1 in various molecular contexts determining essential processes associated with distinct cellular machineries and related to various pathologies, such as cancer, viral infections, neuronal and immunological disorders and CDG.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis

The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

Reduction of Aβ Generation by Schisandrin B through Restraining Beta-Secretase 1 Transcription and Translation.

BackgroundBeta-secretase 1 (BACE1) is a rate-limiting enzyme in the generation of amyloid beta peptides, which are associated with Alzheimer’s disease (AD). It has been reported that Schisandrin B could improve cognitive functions in animal models of AD, but the underlying mechanisms are not completely understood.Material/MethodsIn this research, in order to investigate the effects of Schisandrin B on amyloid-β (Aβ) metabolism and its mechanisms, amyloid precursor protein (APP) and its proteolytic products were determined by enzyme-linked immunosorbent assay (ELISA), western blotting, and RT-PCR after incubation of N2a/Swe cells with Schisandrin B.ResultsThe results indicated that Schisandrin B can significantly reduce the level of secretion of Aβ40 and Aβ42 secreted in N2a/Swe cells. Additionally, there was nonsignificant change in APP level after Schisandrin B treatment. Treatment of Schisandrin B dramatically reduced the mRNA and protein expression levels of BACE1. Moreover, Schisandrin B treatment resulted in a reduction of protein level of sAPPβ, an APP fragment cleavage by BACE1.ConclusionsThese results suggest that Schisandrin B inhibits the transcription and translation of BACE1, suppresses the activity of BACE1, and ultimately attenuates Aβ generation, which provides a novel mechanism for the regulation of Aβ metabolism by Schisandrin B.

Release of Proteolysis Products with ACE-Inhibitory and Antioxidant Activities in Probiotic Yogurt Containing Different Levels of Fat and Prebiotics

In this study, the effects of prebiotic ingredients (inulin and wheat fiber) and fat percentage (0, 2 and 3.5%) on physicochemical properties, degree of proteolysis, antioxidant and ACE-inhibitory activity within 21 days storage of yogurt containing probiotic Lactobacillus casei at 5 ± 1 °C were evaluated. The results of statistical analysis showed that the application of prebiotics led to a significant increase in water-holding capacity, proteolysis and ACE-inhibitory activity of samples. The degree of proteolysis in yogurt increased as storage time elapsed (P < 0.05) but when proteolysis exceeded a certain threshold, this trend began to decline. Also, during storage time, water holding capacity reduced initially but increased thereafter. Moreover, based on our findings, the survival of L. casei, in samples treated with inulin and wheat fiber, increased significantly as compared to the control sample (P < 0.05) whereas the fat percentage did not reflect significant (P < 0.05) effect on the survival of probiotic bacteria. Furthermore, prebiotic ingredients and the presence of probiotic cultures had significant (P < 0.05) effect on the antioxidant activity of samples.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Label-free optical monitoring of proteolytic reaction products using nanoporous silica colloidal assembly

An optical biosensor for rapid monitoring of proteolytic activity is constructed by immobilization of proteases onto multifunctional silica colloidal assembly (SCA). The SCA serves as Fabry-Pérot thin film, which is highly sensitive to the presence of biomolecules (e.g., enzymes, proteins and short peptides) within the nanostructure. Moreover, the SCA acts as a size-exclusion element, allowing to entrap and to concentrate the enzymatic degradation products for downstream mass spectrometry analysis for substrate profiling and cleavage sites identification.

Changes in structure and antioxidant activity of β-lactoglobulin by ultrasound and enzymatic treatment

Effects of ultrasound (20–40% amplitudes at 45–55 °C) and enzymatic (pepsin and trypsin) treatment on structure and antioxidant activity of β-lactoglobulin were studied. Changes in structure of β-lactoglobulin were investigated using spectroscopy techniques and changes in antioxidant activity were measured by chemical and cellular-based assays. Ultrasound treatment had considerable impact on the structure of β-lactoglobulin and increased the susceptibility of β-lactoglobulin to both pepsin and trypsin proteolysis. Intrinsic fluorescence intensity of β-lactoglobulin was increased by ultrasound and then decreased after following enzymatic treatment. Compared with control, the β-lactoglobulin after ultrasound and enzymatic treatments showed significantly higher oxygen scavenging activities in Caco-2 cells models, ABTS (2, 2′-Azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity and oxygen radical absorbance capacity (p < 0.05). Results indicated that ultrasound treatment increased the proteolysis of β-lactoglobulin by both pepsin and trypsin and improved the antioxidant activity of the protein and its proteolytic products.

Proteolysis, microbiology, volatiles and sensory evaluation of Algerian traditional cheese Bouhezza made using goat’s raw milk

ABSTRACTThe objective of this work was to characterize the traditional cheese, Bouhezza made with raw goat’s milk. There were three cheese making performed; two at the laboratory scale (FH1, FH2) and one in a farm (FH3). The evaluation consisted in the determination of physico-chemical characteristics, proteolysis, microbiological and sensory status. The microbiological characterization was performed during cheese ripening with identification of lactic acid bacteria by 16S rDNA sequencing using PCR-TTGE. The sensory analysis and volatile profile were determined by GC-MS. The pH decreased during cheese ripening from 4.69 to 3.99. The moisture in defatted cheese was 79.05% and fat-in-dry matter 34.77%, which allowed its classification according to the Codex alimentarius as ripened soft and mid-fat cheese. The urea-PAGE pattern of caseins showed a fast proteolysis, which started from the first day of ripening. The sensory evaluation showed that Bouhezza cheese was salty, spicy and intense in odour and aroma. A total, of 100 volatile compounds were identified, where the main components were carboxylic acids (14 compounds) followed by their esters (32) and alcohols (13), aldehydes (5), ketones (8), terpenes and miscellaneous compounds. This study showed that Bouhezza cheese was considered as a product of satisfactory microbiological quality and rich in LABs whose contribute to its ripening, where many of identified strains were the same as those found in other dairy products. The main sensory descriptors as flavour and odor, was lactic flavor and lactic acidic odor. The products of proteolysis gives to Bouhezza a riches in volatiles and fragrance characteristics, which reflect the uniqueness of this cheese of terroir. Finally, an identity card of the Bouhezza cheese was achieved following results of this study.

APP/Go protein Gβγ-complex signaling mediates Aβ degeneration and cognitive impairment in Alzheimer's disease models

Deposition of amyloid-β (Aβ), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aβ-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aβ deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aβ toxicity by a mechanism that required Go-Gβγ complex signaling and p38–mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gβγ complex, inhibited Aβ-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aβ pathology. Our data provide further evidence for the involvement of APP/Go protein in Aβ-induced degeneration and reveal that Gβγ complex is a signaling target potentially relevant for developing therapies for halting Aβ degeneration in AD.

Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin.

Many Clostridium perfringens strains produce NanI as their major sialidase. Previous studies showed that NanI could potentiate C. perfringens epsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects on C. perfringens enterotoxin and beta toxin, which are also important toxins for C. perfringens diseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence of C. perfringens to enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor for C. perfringens enteritis and enterotoxemia.