C-terminal Amino Acid Residues Research Articles

Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that Maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrate that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain, and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS are essential for Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates proper folding of nutrient permeases in the ER under the stress condition. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Triphenylphosphonium Analogs of Short Peptide Related to Bactenecin 7 and Oncocin 112 as Antimicrobial Agents.

Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112-Bac(1-10, R/Y)-and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

Expression and characterization of C-terminal truncated mutants of γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1

The gene encoding γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1 was cloned in Escherichia coli. Recombinant PaGGTII showed a weak activity (0.0332 U/mg), and it can be easily inactivated. Multiple alignment of microbial GGTs showed the redundancy of the C-terminal of the small subunit of PaGGTII in length. The truncation of eight amino acid residues at the C-terminal of PaGGTII remarkably improved the activity and stability of the enzyme (PaGGTIIΔ8; 0.388 U/mg). Further truncation at the C-terminal also provided the enzyme relatively higher activity (PaGGTIIΔ9, −Δ10, −Δ11, and −Δ12). Among C-terminal truncated mutants, we focused on PaGGTIIΔ8 and examined the effect of C-terminal amino acid residues on the properties of PaGGTIIΔ8 because the activity of PaGGTII was found to be greatly improved when 8 amino acid residues were truncated. Various mutant enzymes with different C-terminal amino acid residues were constructed. They were expressed in E. coli and purified to homogeneity by ion-exchange chromatography. The properties of PaGGTIIΔ8 and the mutants obtained from mutation at E569 were characterized. Km and kcat of PaGGTIIΔ8 for γ-glutamyl-p-nitroanilide (γ-GpNA) were 8.05 mM and 15.49 s−1, respectively. PaGGTIIΔ8E569Y showed the highest catalytic efficiency for γ-GpNA with a kcat/Km of 12.55 mM−1 s−1. Mg2+, Ca2+, and Mn2+ exhibited positive effects on the catalytic activity for PaGGTIIΔ8 and its ten E569 mutants.

Expression of Xanthorhodopsin in Escherichia coli.

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid-protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties.

A mutant cotton fatty acid desaturase 2-1d allele causes protein mistargeting and altered seed oil composition

BackgroundCotton (Gossypium sp.) has been cultivated for centuries for its spinnable fibers, but its seed oil also possesses untapped economic potential if, improvements could be made to its oleic acid content.ResultsPrevious studies, including those from our laboratory, identified pima accessions containing approximately doubled levels of seed oil oleic acid, compared to standard upland cottonseed oil. Here, the molecular properties of a fatty acid desaturase encoded by a mutant allele identified by genome sequencing in an earlier analysis were analyzed. The mutant sequence is predicted to encode a C-terminally truncated protein lacking nine residues, including a predicted endoplasmic reticulum membrane retrieval motif. We determined that the mutation was caused by a relatively recent movement of a Ty1/copia type retrotransposon that is not found associated with this desaturase gene in other sequenced cotton genomes. The mutant desaturase, along with its repaired isozyme and the wild-type A-subgenome homoeologous protein were expressed in transgenic yeast and stably transformed Arabidopsis plants. All full-length enzymes efficiently converted oleic acid to linoleic acid. The mutant desaturase protein produced only trace amounts of linoleic acid, and only when strongly overexpressed in yeast cells, indicating that the missing C-terminal amino acid residues are not strictly required for enzyme activity, yet are necessary for proper subcellular targeting to the endoplasmic reticulum membrane.ConclusionThese results provide the biochemical underpinning that links a genetic lesion present in a limited group of South American pima cotton accessions and their rare seed oil oleic acid traits. Markers developed to the mutant desaturase allele are currently being used in breeding programs designed to introduce this trait into agronomic upland cotton varieties.

Essential elements of radical pair magnetosensitivity in Drosophila

Many animals use Earth’s magnetic field (also known as the geomagnetic field) for navigation1. The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field2. However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations2–8. Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.

Enzymatic Protein Immobilization on Amino-Functionalized Nanoparticles.

The immobilization of proteins on nanoparticles has received much attention in recent years. Among different approaches, enzymatic protein immobilization shows unique advantages because of its site-specific connection. OaAEP1 is a recently engineered peptide ligase which can specifically recognize an N-terminal GL residue (NH2-Gly-Leu) and a C-terminal NGL amino acid residue (Asn-Gly-Leu-COOH) and ligates them efficiently. Herein, we report OaAEP1-mediated protein immobilization on synthetic magnetic nanoparticles. Our work showed that OaAEP1 could mediate C-terminal site-specific protein immobilization on the amino-functionalized Fe3O4 nanoparticles. Our work demonstrates a new method for site-specific protein immobilization on nanoparticles.

Effect of Disulfide Bridge on the Binding of SARS-CoV-2 Fusion Peptide to Cell Membrane: A Coarse-Grained Study.

In this paper, we present the parameterization of the CAVS coarse-grained (CG) force field for 20 amino acids, and our CG simulations show that the CAVS force field could accurately predict the amino acid tendency of the secondary structure. Then, we used the CAVS force field to investigate the binding of a severe acute respiratory syndrome-associated coronavirus fusion peptide (SARS-CoV-2 FP) to a phospholipid bilayer: a long FP (FP-L) containing 40 amino acids and a short FP (FP-S) containing 26 amino acids. Our CAVS CG simulations displayed that the binding affinity of the FP-L to the bilayer is higher than that of the FP-S. We found that the FP-L interacted more strongly with membrane cholesterol than the FP-S, which should be attributed to the stable helical structure of the FP-L at the C-terminus. In addition, we discovered that the FP-S had one major and two minor membrane-bound states, in agreement with previous all-atom molecular dynamics (MD) studies. However, we found that both the C-terminal and N-terminal amino acid residues of the FP-L can strongly interact with the bilayer membrane. Furthermore, we found that the disulfide bond formed between Cys840 and Cys851 stabilized the helices of the FP-L at the C-terminus, enhancing the interaction between the FP-L and the bilayer membrane. Our work indicates that the stable helical structure is crucial for binding the SARS-CoV-2 FP to cell membranes. In particular, the helical stability of FP should have a significant influence on the FP–membrane binding.

P.157 Dominant HSPB6 mutation in a myopathy patient

HSPB6 (also known as Hsp20) is a small heat-shock protein (sHSP) showing highest expression is skeletal, cardiac and smooth muscles. In contrast to most sHSPs, HSPB6 does not readily assemble into large oligomers on its own. However, it heterodimerizes with HSPB1 and hetero-oligomerizes with HSPB1 and αB-crystallin (CRYAB, HSPB5), modulating their oligomer size and chaperone activity. HSPB6 may also be involved in BAG3-mediated autophagy. Mutations in several sHSPs lead to neuromuscular disease. While rare missense variants in <i>HSPB6</i> have been associated with cardiomyopathy, the gene has not been previously linked to skeletal muscle disease. We describe the likely pathogenic heterozygous <i>HSPB6</i> variant c.464delC (p.Pro155Argfs*25) in a 62-year-old man with onset of the disease at 43 years of age characterized by a proximal lower limb weakness and a respiratory insufficiency. At 60 years of age, he walks with 2 canes. Of note, he underwent a cataract surgery at 28 years of age. The single-nucleotide deletion causes a frameshift predicted to replace of the six C-terminal amino acid residues of the HSPB6 protein with 24 erroneous residues. Western blotting of patient biopsy showed the normal and extended HSPB6 proteins at a 50:50 ratio, indicating that the mutant protein is expressed and stable. Histological findings included infrequent central HSPB6 accumulation in the myofibers, and myotilin-positive cytoplasmic bodies. In cell culture experiments, mutant HSPB6 showed reduced solubility compared to the wild-type protein and affected the solubility of coexpressed wild-type HSPB6, HSPB1 and CRYAB. Immunofluorescence microscopy revealed altered CRYAB localization patterns in cells expressing mutant HSPB6. Our results suggest that the <i>HSPB6</i> c.464delC (p.Pro155Argfs*25) mutation may cause muscle disease through a gain-of-function mechanism interfering with the sHSP machinery.

Context-based sensing of orthosomycin antibiotics by the translating ribosome.

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.

Molecular insights into the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG: A molecular dynamics simulation study

Aβ peptide aggregates were the main targets to treat AD. Previous experimental study demonstrated that the 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucopyranose (PGG, containing 5 gallate moieties) dramatically destabilized the preformed Aβ fibril in vitro and in vivo. However, the mechanisms were still poorly understood. Herein, by performing molecular dynamics simulations, the detailed mechanisms of the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG were explored. It was demonstrated that the binding of PGG molecules to Aβ1-42 pentameric protofibril disrupted the β-sheet structure, weakened the interactions between Chain-1 and Chain-2, and disturbed the Lys28-Ala42 salt bridges in Aβ1-42 pentameric protofibril. We found that the hydrophobic interactions, hydrogen bonds, pi-pi interactions and cation-pi interactions existed between PGG molecules and amino acid residues in the corner of L-shaped part (Phe4-Leu17) were conducive to the disaggregation of the close interactions between Chain-1 and Chain-2. Moreover, PGG molecules disrupted the Lys28-Ala42 salt bridges not only by the hydrogen bonds and cation-pi interactions with Lys28 but also through the interactions with C-terminal hydrophobic amino acid residues. We believed that our results could provide significant insights into the mechanisms of the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG.

Multiple Phosphorylations of SR Protein SRSF3 and Its Binding to m6A Reader YTHDC1 in Human Cells.

N6-methyladenosine (m6A) is a well-known RNA modification and has various functions with its binding proteins. Nuclear m6A reader protein YTHDC1 plays a significant role in RNA metabolism including some non-coding RNA such as LINE or circRNA. It is also known to regulate mRNA splicing through recruiting SRSF3 to the targeted mRNAs, which then mediates export of YTHDC1-bound RNA to the cytoplasm. Additionally, it has been indicated that SRSF3 binding to YHTDC1 may be mediated by its dephosphorylated status. However, their binding mechanism, including the positions of dephosphorylated residues of SRSF3, has not been sufficiently investigated. Thus, we explored the mechanism of interaction between SRSF3 and YTHDC1 in human cells. We used co-immunoprecipitation to examine the binding of YTHDC1/SRSF3 through their N- and C-terminal amino-acid residues. Furthermore, dephosphorylation-mimic serine to alanine mutants of SRSF3 indicated the position of phosphorylated residues. Cumulatively, our results demonstrate that YTHDC1 binding to SRSF3 is regulated by not only hypo-phosphorylated residues of arginine/serine-rich (RS) domain of SRSF3 but also other parts of SRSF3 via YTHDC1 N- or C-terminal residues. Our results contribute to the understanding of the complex mechanism of binding between SR protein SRSF3 and the m6A reader YTHDC1 to regulate the expression of mRNA and non-coding RNAs.

The new effector AbSCP1 of foliar nematode (Aphelenchoides besseyi) is required for parasitism rice

Plant parasitic nematodes secrete effector proteins to parasitize hosts successfully. Of these proteins, serine carboxypeptidases have critical roles in pathogenicity. This study investigated the role of new effector AbSCP1 in Aphelenchoides besseyi pathogenicity. In situ hybridization and qRT-PCR analyses indicated that AbSCP1 was exclusively expressed in the esophageal glands and upregulated in juveniles. Subcellular localization assays indicated that the protein was expressed in the nucleus. The ability to hydrolyze C-terminal amino acid residues was proven for AbSCP1. Moreover, RNAi significantly reduced the expression of AbSCP1 and RNAi-treated nematodes’ reproductive potential. Pathogenicity assays on rice showed that RNAi-treated nematodes were less pathogenic than the untreated control groups. These results suggest the important role of AbSCP1 in the A. besseyi infection process.

Influence of the Dynamically Disordered N-Terminal Tail Domain on the Amyloid Core Structure of Human Y145Stop Prion Protein Fibrils.

The Y145Stop mutant of human prion protein (huPrP23-144) is associated with a familial prionopathy and provides a convenient in vitro model for investigating amyloid strains and cross-seeding barriers. huPrP23-144 fibrils feature a compact and relatively rigid parallel in-register β-sheet amyloid core spanning ∼30 C-terminal amino acid residues (∼112–141) and a large ∼90-residue dynamically disordered N-terminal tail domain. Here, we systematically evaluate the influence of this dynamic domain on the structure adopted by the huPrP23-144 amyloid core region, by investigating using magic-angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy a series of fibril samples formed by huPrP23-144 variants corresponding to deletions of large segments of the N-terminal tail. We find that deletion of the bulk of the N-terminal tail, up to residue 98, yields amyloid fibrils with native-like huPrP23-144 core structure. Interestingly, deletion of additional flexible residues in the stretch 99–106 located outside of the amyloid core yields shorter heterogenous fibrils with fingerprint NMR spectra that are clearly distinct from those for full-length huPrP23-144, suggestive of the onset of perturbations to the native structure and degree of molecular ordering for the core residues. For the deletion variant missing residues 99–106 we show that native huPrP23-144 core structure can be “restored” by seeding the fibril growth with preformed full-length huPrP23-144 fibrils.

Antibacterial mechanism of the Asp-Asp-Asp-Tyr peptide

Previously, we found that ASP-ASP-ASP-TYR (DDDY) from Dendrobium aphyllum has a minimum inhibitory concentration of 36.15 mg/mL against Pseudomonas aeruginosa. Here, we explored the antibacterial mechanism of DDDY and its potential preservation applications. Metabolomic and transcriptomic analyses revealed that DDDY mainly affects genes involved in P. aeruginosa membrane transport and amino acid metabolism pathways. Molecular dynamics simulation revealed that DDDY had a stronger effect on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine phospholipid membranes than on 1-palmitoyl-2-oleoyl-lecithin or 1-palmitoyl-2-oleoyl phosphatidylglycerol membranes, with high DDDY concentrations displaying stronger efficacy on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. Mechanistically, the N-terminal of DDDY first bound to the phospholipid head group, while its C-terminal amino acid residue bound the hydrophobic tail, thereby creating a gap in the membrane when the phospholipids were clustered by hydrogen bonding. Finally, DDDY inhibited the growth of food microorganisms inoculated onto chestnut kernels, suggesting that DDDY is a promising antibacterial agent against multidrug-resistant gram-negative bacteria.

Side-Chain Anchoring Strategies for the Synthesis of Peptide Thioesters and Selenoesters.

Peptides bearing C-terminal thioester and selenoester functionalities are essential precursors for the chemical synthesis of larger proteins using ligation chemistry, including native chemical ligation (NCL) and diselenide-selenoester ligation (DSL). The use of a side-chain anchoring thioesterification or selenoesterification approach offers a robust method to access peptide thioesters or peptide selenoesters in excellent yields and in high purity. Importantly, this methodology overcomes solubility issues and epimerization of the C-terminal amino acid residue that can occur using solution-phase approaches. Detailed methods for the solid-phase synthesis of peptide thioesters and selenoesters using a side-chain anchoring approach are outlined in this article.

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics.

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

Direct Evidence for Viral Antigen Presentation during Latent Cytomegalovirus Infection.

Murine models of cytomegalovirus (CMV) infection have revealed an immunological phenomenon known as “memory inflation” (MI). After a peak of a primary CD8+ T-cell response, the pool of epitope-specific cells contracts in parallel to the resolution of productive infection and the establishment of a latent infection, referred to as “latency.” CMV latency is associated with an increase in the number of cells specific for certain viral epitopes over time. The inflationary subset was identified as effector-memory T cells (iTEM) characterized by the cell surface phenotype KLRG1+CD127−CD62L−. As we have shown recently, latent viral genomes are not transcriptionally silent. Rather, viral genes are sporadically desilenced in a stochastic fashion. The current hypothesis proposes MI to be driven by presented viral antigenic peptides encoded by the corresponding, stochastically expressed viral genes. Although this mechanism suggests itself, independent evidence for antigen presentation during viral latency is pending. Here we fill this gap by showing that T cell-receptor transgenic OT-I cells that are specific for peptide SIINFEKL proliferate upon adoptive cell transfer in C57BL/6 recipients latently infected with murine CMV encoding SIINFEKL (mCMV-SIINFEKL), but not in those latently infected with mCMV-SIINFEKA, in which antigenicity is lost by mutation L8A of the C-terminal amino acid residue.