Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis.

Cholesterol dyshomeostasis has been linked to the pathogenesis of sporadic Alzheimer's disease (AD). In furthering the understanding of mechanisms by which increased levels of circulating cholesterol augments the risk of developing sporadic AD, others and we have reported that low-density lipoprotein (LDL) enters brain parenchyma by disrupting the blood-brain barrier and that endolysosome de-acidification plays a role in LDL-induced amyloidogenesis in neurons. Here, we tested the hypothesis that endolysosome de-acidification was central to amyloid-ß (Aß) generation and that acidifying endolysosomes protects against LDL-induced increases in Aß levels in neurons. We demonstrated that LDL, but not HDL, de-acidified endolysosomes and increased intraneuronal and secreted levels of Aß. ML-SA1, an agonist of endolysosome-resident TRPML1 channels, acidified endolysosomes, and TRPML1 knockdown attenuated ML-SA1-induced endolysosome acidification. ML-SA1 blocked LDL-induced increases in intraneuronal and secreted levels of Aß as well as Aß accumulation in endolysosomes, prevented BACE1 accumulation in endolysosomes, and decreased BACE1 activity levels. LDL downregulated TRPML1 protein levels, and TRPML1 knockdown worsens LDL-induced increases in Aß. Our findings suggest that endolysosome acidification by activating TRPML1 may represent a protective strategy against sporadic AD.

Activation Stoichiometry and Pore Architecture of TRPA1 Probed with Channel Concatemers.

The nociceptor ion channel TRPA1 detects a wide range of hazardous chemicals, including reactive electrophiles such as cinnamaldehyde, which gate the channel allowing Na+ and Ca2+ entry. TRPA1 assembles as a tetramer, with a central pore within which an aspartate residue (D918) determines Ca2+ permeability. Here, we report that introduction of histidine at this position, D918H, makes TRPA1 channels sensitive to block by nanomolar concentration of Zn2+ and can be used to functionally tag subunits in concatemers. Concatemers with increasing numbers of D918H subunits display increasing sensitivity to Zn2+ inhibition, indicating that the four side chains at position 918 of the tetramer directly coordinate Zn2+ and other permeating divalent cations. In the published structure of TRPA1, this requires a rearrangement of the pore region which may represent the true open state of the channel. Concatemeric channels containing subunits mutated to be insensitive to reactive electrophiles (C622S) could be activated by cinnamaldehyde when as few as two subunits contained intact ligand binding sites. Activation upon liganding of just two of the four possible subunits may represent an optimal strategy to rapidly and reliably detect noxious chemicals.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

lspA gene of Mycobacterium tuberculosis co-transcribes with Rv1540 and induced by surface and acidic stress.

Lipoprotein signal peptidase, lspA (Rv1539), is the only known gene in mycobacterial genome for cleaving the signal sequence from prolipoprotein to form mature lipoprotein. It has been implicated in maintaining the virulence of Mycobacterium tuberculosis. The regulation of lspA had not been studied so far. Here, we identify a novel operon lspA-Rv1540 in M. tuberculosis. We detected co-transcription of the open reading frames of lspA-Rv1540 in in-vitro as well as in ex-vivo conditions. Analysis of the sequence upstream to lspA revealed a strong promoter activity that was shown to be induced significantly by surface stress and acidic environment.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Characterization of an aspartate-dependent acid survival system in Yersinia pseudotuberculosis.

Enteric bacteria have developed various survival systems that protect against acid stress. In this study, an aspartate-dependent acid survival system is characterized in Yersinia pseudotuberculosis. The expression of aspartase (AspA) was confirmed to be increased at acidic pH by proteomic and lacZ fusion analyses. Addition of aspartate increased acid survival of the wild type but not the aspA knockout mutant. AspA increases acid survival by producing ammonia as demonstrated by mutation and in vitro enzyme activity analyses. This is the first demonstration that an enzyme involved in aspartate metabolism plays a role in acid survival in an enteric bacterium.

The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His-Asp phosphorelay RcsCDB/AF.

The RcsCDB signal transduction system is an atypical His-Asp phosphorelay. Notably, the response regulator RcsB can be activated either by phosphorylation through the RcsCD pathway or by an accessory cofactor RcsA. Although conserved in Enterobacteriaceae, the role of this system in adaptation to environmental stress conditions is largely unknown. This study reveals that the response regulator RcsB is essential to glutamate-dependent acid resistance, a condition pertinent to the lifestyle of Escherichia coli. The requirement for RcsB is independent of its activation by either the RcsCD or the RcsA pathway. The basal activity of RcsB appears to be necessary and sufficient for acid resistance. The sensitivity of the rcsB strain to low pH is correlated to a strong reduction of the expression of the glutamate decarboxylase genes, gadA and gadB, during the stationary phase of growth. This effect on gadA/B expression is not mediated by the general stress sigma factor RpoS, but does require a functional gadE allele and the previously identified GadE box. Therefore activation of gadAB expression and acid resistance absolutely requires both GadE and RcsB. In contrast, an increase in RcsB activity through the activation of the RcsCD phosphorelay or the RcsA pathway or through overproduction of the protein leads to general repression of the expression of the gad genes and a corresponding reduction in acid resistance.

Requirement of the proteasome for the trimming of signal peptide-derived epitopes presented by the nonclassical major histocompatibility complex class I molecule HLA-E.

The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.