Production and Purification of Novel Hypocholesterolemic Peptides from Lactic Fermented Spirulina platensis through High Hydrostatic Pressure-Assisted Protease Hydrolysis

This research focuses on the proteolytic capacity of Spirulina platensis and their hypocholesterolemic activity via the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) inhibitory activity. To select suitable proteases for releasing peptides with high HMGR-inhibiting activity from S. platensis, eight commonly used commercial proteases were used in protease hydrolysis under high hydrostatic pressure (HHP, 100 MPa or 0.1 MPa) at 50 °C for 24 h. The Peptidase R group had the highest inhibitory capacity (67%). First, S. platensis was fermented with seven mixed lactic acid bacteria for 5 h at 42 °C. This was followed by the addition of Peptidase R under high hydrostatic pressure (100 MPa at 50 °C) for 0–6 h of enzymatic hydrolysis (HHP-FH-PR6) to determine the hydrolytic capacity of S. platensis protein. As the hydrolysis time extended to 6 h, the peptide content increased from 96.8 mg/mL to 339.8 mg/mL, and the free amino acid content increased from 24 mg/mL to 115.2 mg/mL, while inhibition of HMGR increased from 67.0% to 78.4%. In an experimental simulation of in vitro gastrointestinal digestion, the IC50 of HHP-FH-PR6G on HMGR was 3.5 μg peptide/mL. Peptides with inhibitory activity on HMGR were purified, and their sequences were identified as Arg-Cys-Asp and Ser-Asn-Val (IC50: 6.9 and 20.1 μM, respectively).

The JAK2-STAT3 pathway controls a beneficial proteostasis response of reactive astrocytes in Huntington’s disease

Huntington’s disease (HD) is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin (mHTT) and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in HD that involves morphological, molecular and functional changes. How reactive astrocytes contribute to HD is still an open question, especially because their reactive state is poorly reproduced in mouse models.Here, we show that the JAK2-STAT3 pathway, a central cascade controlling the reactive response of astrocytes, is activated in the putamen of HD patients. Selective activation of this cascade in astrocytes reduces the number and size of neuronal mHTT aggregates and improves neuronal features in two HD mouse models. Moreover, activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomes and the ubiquitin-proteasome system, and enhances their production of the co-chaperone DNAJB1, which is released in exosomes.Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in HD, which involves bi-directional signalling with neurons to reduce mHTT aggregation and toxicity.

INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions

Despite an increased understanding of leukemogenesis, specific mechanisms that underlie stemness in leukemia remain largely undefined. Here, we report a novel pathway which regulates leukemic differentiation through control of lysosomal biology. We show that disruption of INPP4B results in dysregulated lysosomal gene networks, reduced lysosomal numbers and proteolytic capacity in leukemia. Inpp4b-deficient HSCs and LSCs are functionally compromised. Inpp4b-deficient leukemia models develop more differentiated leukemias with reduced disease initiating potential, and improved overall survival compared to Inpp4b-expressing leukemias. Together, our data is consistent with a model where INPP4B restricts differentiation of LSCs through regulation of lysosomal function. These data provide a mechanism to explain the association of INPP4B with aggressive AML and highlight avenues for LSC-specific leukemia therapies.

Yersinia pestis Plasminogen Activator

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Degradation of Lon in Caulobacter crescentus

ABSTRACT Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell. IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

Sulforaphane-cysteine inhibited migration and invasion via enhancing mitophagosome fusion to lysosome in human glioblastoma cells

Here we uncovered the involved subcellular mechanisms that sulforaphane-cysteine (SFN-Cys) inhibited invasion in human glioblastoma (GBM). SFN-Cys significantly upregulated 45 and downregulated 14 microtubule-, mitophagy-, and invasion-associated proteins in GBM cells via HPLC–MS/MS and GEO ontology analysis; SFN-Cys disrupted microtubule by ERK1/2 phosphorylation-mediated downregulation of α-tubulin and Stathmin-1 leading to the inhibition of cell migration and invasion; SFN-Cys downregulated invasion-associated Claudin-5 and S100A4, and decreased the interaction of α-tubulin to Claudin-5. Knockdown of Claudin-5 and S100A4 significantly reduced the migration and invasion. Besides, SFN-Cys lowered the expressions of α-tubulin-mediated mitophagy-associated proteins Bnip3 and Nix. Transmission electron microscopy showed more membrane-deficient mitochondria and accumulated mitophagosomes in GBM cells, and mitochondria fusion might be downregulated because that SFN-Cys downregulated mitochondrial fusion protein OPA1. SFN-Cys increased the colocalization and interplay of LC3 to lysosomal membrane-associated protein LAMP1, aggravating the fusion of mitophagosome to lysosome. Nevertheless, SFN-Cys inhibited the lysosomal proteolytic capacity causing LC3II/LC3I elevation but autophagy substrate SQSTM1/p62 was not changed, mitophagosome accumulation, and the inhibition of migration and invasion in GBM cells. These results will help us develop high-efficiency and low-toxicity anticancer drugs to inhibit migration and invasion in GBM.

Degradation of Lon in Caulobacter crescentus

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about post-translational control of these critical machines. In this study we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates such as the replication initiator DnaA to lethally low levels. Taken together, this work demonstrates a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.ImportanceProteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

Impact of Fermentation on the Recovery of Antioxidant Bioactive Compounds from Sea Bass Byproducts

The aim of the present research was to obtain antioxidant compounds through the fermentation of fish byproducts by bacteria isolated from sea bass viscera. To that purpose, bacteria from sea bass stomach, intestine, and colon were isolated. With the selected bacteria, growing research was undertaken, fermenting different broths prepared with sea bass meat or byproducts. After the fermentation, the antioxidant activity, phenolic acids, and some proteins were evaluated. The main phenolic acids obtained were DL-3-phenyl-lactic acid and benzoic acid at a maximum concentration of 466 and 314 ppb, respectively. The best antioxidant activity was found in the extracts obtained after the fermentation of fish byproducts broth by bacteria isolated from the colon (6502 μM TE) and stomach (4797 μM TE). Moreover, a positive correlation was found between phenolic acids obtained after the fermentation process and the antioxidant activity of the samples. It was also concluded that the lactic acid bacteria isolated from sea bass had an important proteolytic capacity and were able to synthesize phenolic acids with antioxidant capacity. This work has shown the relevance of fermentation as a useful tool to valorize fish byproducts, giving them an added economic value and reducing their environmental impact.

The transfer of specific mitochondrial lipids and proteins to lipid droplets contributes to proteostasis upon stress and aging in the eukaryotic model system Saccharomyces cerevisiae

Originally Lipid droplets (LDs) were considered as being droplets for lipid storage only. Increasing evidence, however, demonstrates that LDs fulfill a pleiotropy of additional functions. Among them is the modulation of protein as well as lipid homeostasis. Under unfavorable pro-oxidative conditions, proteins can form aggregates which may exceed the overall proteolytic capacity of the proteasome. After stress termination LDs can adjust and support the removal of these aggregates. Additionally, LDs interact with mitochondria, specifically take over certain proteins and thus prevent apoptosis. LDs, which are loaded with these harmful proteins, are subsequently eliminated via lipophagy. Recently it was demonstrated that this autophagic process is a modulator of longevity. LDs do not only eliminate potentially dangerous proteins, but they are also able to prevent lipotoxicity by storing specific lipids. In the present study we used the model organism Saccharomyces cerevisiae to compare the proteome as well as lipidome of mitochondria and LDs under different conditions: replicative aging, stress and apoptosis. In this context we found an accumulation of proteins at LDs, supporting the role of LDs in proteostasis. Additionally, the composition of main lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols, triacylglycerols, ceramides, phosphatidic acids and ergosterol of LDs and mitochondria changed during stress conditions and aging.