Molecular chaperone ability to inhibit amyloid-derived neurotoxicity, but not amorphous protein aggregation, depends on a conserved pH-sensitive Asp residue

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but the respective mechanisms are not fully understood. The BRICHOS domain constitutes a disease-associated small heat shock protein-like chaperone family, with activities against both amyloid toxicity and amorphous protein aggregation. Here, we show that the activity of two BRICHOS domain families against Alzheimer’s disease associated amyloid-β neurotoxicity to mouse hippocampi in vitro depends on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The conserved Asp in its ionized state promotes structural flexibility of the BRICHOS domain and has a pKa value between pH 6.0–7.0, suggesting that chaperone effects against amyloid toxicity can be affected by physiological pH variations. Finally, the Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families and animal species, indicating independent evolution of molecular chaperone activities against amyloid fibril formation and non-fibrillar amorphous protein aggregation.

Evolutionary expansion of poly-aspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen orthologs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased autoactivation of T7 trypsinogen 3-fold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side-chains are preferred for maximal autoactivation and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by N-terminal truncation with chymotrypsin C reduced autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable to that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the poly-aspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66–80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66–80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact β-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2′-O-methyl transfer by SARS-CoV-2 nsp16

Capping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2′-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.

Ygr125w/Vsb1-dependent accumulation of basic amino acids into vacuoles of Saccharomyces cerevisiae

The Ygr125w was previously identified as a vacuolar membrane protein by a proteomic analysis. We found that vacuolar levels of basic amino acids drastically decreased in ygr125wΔ cells. Since N- or C-terminally tagged Ygr125w was not functional, an expression plasmid of YGR125w with HA3-tag inserted in its N-terminal hydrophilic region was constructed. Introduction of this plasmid into ygr125w∆ cells restored the vacuolar levels of basic amino acids. We successfully detected the uptake activity of arginine by the vacuolar membrane vesicles depending on HA3-YGR125w expression. A conserved aspartate residue in the predicted first transmembrane helix (D223) was indispensable for the accumulation of basic amino acids. YGR125w has been recently reported as a gene involved in vacuolar storage of arginine; and it is designated as VSB1. Taken together, our findings indicate that Ygr125w/Vsb1 contributes to the uptake of arginine into vacuoles and vacuolar compartmentalization of basic amino acids.

Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants

Posttranscriptional modification of tRNA is critical for efficient protein translation and proper cell growth, and defects in tRNA modifications are often associated with human disease. Although most of the enzymes required for eukaryotic tRNA modifications are known, many of these enzymes have not been identified and characterized in several model multicellular eukaryotes. Here, we present two related approaches to identify the genes required for tRNA modifications in multicellular organisms using primer extension assays with fluorescent oligonucleotides. To demonstrate the utility of these approaches we first use expression of exogenous genes in yeast to experimentally identify two TRM1 orthologs capable of forming N2,N2-dimethylguanosine (m2,2G) on residue 26 of cytosolic tRNA in the model plant Arabidopsis thaliana. We also show that a predicted catalytic aspartate residue is required for function in each of the proteins. We next use RNA interference in cultured Drosophila melanogaster cells to identify the gene required for m2,2G26 formation on cytosolic tRNA. Additionally, using these approaches we experimentally identify D. melanogaster gene CG10050 as the corresponding ortholog of human DTWD2, which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp3U) on residue 20a of cytosolic tRNA. We further show that A. thaliana gene AT2G41750 can form acp3U20b on an A. thaliana tRNA expressed in yeast cells, and that the aspartate and tryptophan residues in the DXTW motif of this protein are required for modification activity. These results demonstrate that these approaches can be used to study tRNA modification enzymes.

A Simplified Method of Synthesis to Obtain Zwitterionic Cellulose under Mild Conditions with Active Ionic Moieties

A simplified procedure to synthesize zwitterionic cellulose by means of N-protected aspartic anhydride under mild conditions was developed. The preparation of modified cellulose samples was carried out under heterogeneous, aqueous conditions by reacting NH4OH-activated cellulose with aspartic anhydrides N-protected with trifluoroacetyl (TFAc) and carbobenzyloxy (Cbz). Modified cellulose samples Cel-Asp-N-TFAc and Cel-Asp-N-Cbz were characterized by Fourier Transform Infrared (FTIR) and 13C solid state Nuclear Magnetic Resonance (NMR) spectroscopy. The functionalization degree of each cellulose sample was determined by the 13C NMR signal integration values corresponding to the cellulose C1 vs. the Cα of the aspartate residue and corroborated by elemental analysis. In agreement, both analytical methods averaged a grafting degree of 20% for Cel-Asp-N-TFAc and 16% for Cel-Asp-N-Cbz. Conveniently, Cel-Asp-N-TFAc was concomitantly partially N-deprotected (65%) as determined by the ninhydrin method. The zwitterion character of this sample was confirmed by a potentiometric titration curve and the availability of these amino acid residues on the cellulose was inspected by adsorption kinetics method with a 100 mg L−1 cotton blue dye solution. In addition, the synthesis reported in the present work involves environmentally related advantages over previous methodologies developed in our group concerning to zwitterionic cellulose preparation.