Identification of Residues Critical for FPR2 Activation by the Cryptic Peptide Mitocryptide-2 Originating from the Mitochondrial DNA-Encoded Cytochrome b.

Similar to bacteria, synthesis of mitochondrial DNA-encoded proteins requires an N-formylated methionine to initiate translation. Thus, the N-formylated methionine peptides originating from mitochondria should be recognized as danger signals. To date, only one such peptide, denoted as mitocryptide-2 (MCT-2), originating from the N-terminal of the mitochondrial cytochrome b, has been isolated from mammalian tissues. Human neutrophils express FPR1 and FPR2 that detect formyl peptides, and the precise structural determinants for receptor recognition remain to be elucidated. MCT-2 is known to activate neutrophils through FPR2 but not FPR1. The aim of this study was to elucidate the structural determinants of importance for receptor preference and human neutrophil activation in MCT-2 by generating a series of MCT-2 variants. We show that there is an absolute requirement for the N-formyl group and the side chain of Met1 at position 1 of MCT-2 but also the C terminus is of importance for MCT-2 activity. We also uncovered individual side chains that positively contribute to MCT-2 activity as well as those suppressed in the response. The MCT-2 peptide and its two polymorphic variants ([Thr7]MCT-2 and [Ser8]MCT-2) all activated neutrophils, but MCT-2 containing Ile7 and Asn8 was the most potent. We also show that some peptide variants displayed a biased FPR2-signaling property related to NADPH oxidase activation and ß-arrestin recruitment, respectively. In conclusion, we disclose several critical elements in MCT-2 that are required for neutrophil activation and disclose structural insights into how FPR2 recognition of this mitochondrial DNA-derived peptide may increase our understanding of the role of FPR2 in aseptic inflammation.

Mitochondrial protein-derived cryptides: Are endogenous N-formylated peptides including mitocryptide-2 components of mitochondrial damage-associated molecular patterns?

Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also ß-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.

Infección por Trypanosoma cruzi y polimorfismo del Citocromo B del ADN mitocondrial en Triatoma maculata de Anzoategui y Portuguesa, Venezuela / Trypanosoma cruzi infection and cytochrome b polimorphism of mitochondrial dna in Triatoma maculata of Anzoátegui and Portuguesa states, Venezuela

Con el fin de entender la dinámica poblacional de Triatoma maculata, se analizó el polimorfismo genético y los índices de infección con Trypanosoma cruzi, utilizando triatominos provenientes de ecotopos y regiones geográficas diferentes. El índice de infección parasitaria para T. maculata, fue de 29.8% a través de la observación directa al microscopio y 40.3% utilizando el método de reacción en cadena de la polimerasa. Los niveles de infección encontrados incrementan la importancia de T. maculata como vector involucrado en el ciclo de transmisión de T. cruzi. El análisis del polimorfismo de longitud de fragmentos de restricción de una región del gen Cyt B, permitió establecer en forma preliminar, diferencias en los patrones de bandas de este gen, según el origen geográfico de cada población. Esto puede asociarse a cambios relacionados con procesos adaptativos involucrados en la colonización de nuevos hábitats. No se observó variación genética para vectores capturados en diferentes ecotopos de una misma localidad. Sin embargo es evidente la participación del vector en el ciclo de transmisión, mostrando que la presencia de T. maculata en las casas no puede ser ignorada

In order to understand more about the populational dynamics of Triatoma maculata, the genetic polimorphism and the infection indexes of Trypanosoma cruzi were analysed, using triatomine obtained from different ecotopes and geographical regions. The parasitic infection index of T. maculata was 29.8% using the microscope direct observation, and 40.3% by the polymerase chain reaction method. Both methods were important for epidemiological screening of the vectors with low potential of infection. The amplification of one region the Cyt B gene of these organisms, followed by a restriction fragments length polymorphism analysis, allowed us to establish different patterns of bands according to the geographic origin of each population, which indicates the lack of migration between individuals of Portuguesa and Anzoátegui states. These genetic differences may be associated with changes in adaptative events involved in the colonization of new habitats. The lack of polymorphism among vectors collected in different habitats of the same region showed an important genetic flow which has epidemiological implications in the risk of transmission of the disease, showing that the presence of T. maculata in houses cannot be ignored

CD4+ T-cell response to mitochondrial cytochrome B in human melanoma.

Mitochondrial DNA (mtDNA) is highly susceptible to mutations due to the low level of DNA repair and the presence of a high level of reactive oxygen species in the organelle. Although mtDNA mutations have been implicated in degenerating diseases, aging, and cancer, very little is known about the role of T cells in immunosurveillance for mtDNA aberrations. Here, we describe T-cell recognition of a peptide translated from an alternative open reading frame of the mitochondrial cytochrome b (cyt b) gene in melanoma cells established from a patient. To understand how the cyt b gene is transcribed and translated in tumor cells, we found that cyt b-specific CD4(+) T cells only recognized protein fractions derived from cytoplasm and not from mitochondria. However, T-cell recognition of tumor cells could be inhibited by treatment of tumor cells with rhodamine 6G inhibitor, which depletes mitochondria. These findings suggest that cyt b mRNA is leaked out of the mitochondria and then translated in the cytoplasm for presentation to CD4(+) T cells. The cyt b cDNAs from this patient contain highly heteroplasmic transition mutations compared with control cell lines, suggesting a compromise of mitochondrial integrity that may have contributed to melanoma induction or progression. These findings provide the first example of a mitochondrial immune target for CD4(+) T cells and therefore have implications for the immunosurveillance of mitochondrial aberrations in cancer patients.

Identification of Pseudomonas aeruginosa-induced genes in human mast cells using suppression subtractive hybridization: up-regulation of IL-8 and CCL4 production.

Mast cells have recently been found to be a major player in the host defence against bacterial infection through secretion of potent mediators. Identification of bacteria-induced mast cell mediators and intracellular signalling molecules involved during bacterial infection remains a major area of investigation. Recently we found an active interaction between mast cells and Pseudomonas aeruginosa bacteria. To further characterize specific genes in mast cells modulated by P. aeruginosa, we used a new approach for the study of mast cell-bacteria interaction; the suppression subtractive hybridization (SSH). SSH approach does not require a prerequisite knowledge of target genes and does not rely on the availability of the assay reagents for the specific genes. Using SSH, 94 clones were randomly selected from the subtracted cDNA library for differential screening leading to the identification of 14 P. aeruginosa-up-regulated transcripts. Sequence analysis revealed that expression of IL-1, IL-8 and CCL4 was increased by human mast cells after P. aeruginosa infection. Increased production of IL-1, IL-8 and CCL4 was confirmed at the protein levels. In addition, sequence analysis of the clones also suggests that ribosomal protein S3 and cytochrome b as well as additional 4 uncharacterized genes may potentially be involved in P. aeruginosa pathogenesis. Thus, SSH is an effective approach by identifying potential molecular targets for the study of mechanisms involved in P. aeruginosa and mast cell interaction.

Affinity purification of antibodies by using Ni2+-resins on which inclusion body-forming proteins are immobilized.

Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound.