Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Circulating N-formylmethionine and metabolic shift in critical illness: a multicohort metabolomics study.

BACKGROUND: Cell stress promotes degradation of mitochondria which release danger-associated molecular patterns that are catabolized to N-formylmethionine. We hypothesized that in critically ill adults, the response to N-formylmethionine is associated with increases in metabolomic shift-related metabolites and increases in 28-day mortality. METHODS: We performed metabolomics analyses on plasma from the 428-subject Correction of Vitamin D Deficiency in Critically Ill Patients trial (VITdAL-ICU) cohort and the 90-subject Brigham and Women's Hospital Registry of Critical Illness (RoCI) cohort. In the VITdAL-ICU cohort, we analyzed 983 metabolites at Intensive Care Unit (ICU) admission, day 3, and 7. In the RoCI cohort, we analyzed 411 metabolites at ICU admission. The association between N-formylmethionine and mortality was determined by adjusted logistic regression. The relationship between individual metabolites and N-formylmethionine abundance was assessed with false discovery rate correction via linear regression, linear mixed-effects, and Gaussian graphical models. RESULTS: Patients with the top quartile of N-formylmethionine abundance at ICU admission had a significantly higher adjusted odds of 28-day mortality in the VITdAL-ICU (OR, 2.4; 95%CI 1.5-4.0; P = 0.001) and RoCI cohorts (OR, 5.1; 95%CI 1.4-18.7; P = 0.015). Adjusted linear regression shows that with increases in N-formylmethionine abundance at ICU admission, 55 metabolites have significant differences common to both the VITdAL-ICU and RoCI cohorts. With increased N-formylmethionine abundance, both cohorts had elevations in individual short-chain acylcarnitine, branched chain amino acid, kynurenine pathway, and pentose phosphate pathway metabolites. CONCLUSIONS: The results indicate that circulating N-formylmethionine promotes a metabolic shift with heightened mortality that involves incomplete mitochondrial fatty acid oxidation, increased branched chain amino acid metabolism, and activation of the pentose phosphate pathway.

Pseudouridine and N-formylmethionine associate with left ventricular mass index: Metabolome-wide association analysis of cardiac remodeling.

BACKGROUND: Heart failure (HF) is the fastest growing form of cardiovascular disease both nationally and globally, underlining a need to phenotype subclinical HF intermediaries to improve primary prevention. OBJECTIVES: We aimed to identify novel metabolite associations with left ventricular (LV) remodeling, one upstream HF intermediary, among a community-based cohort of individuals. METHODS: We examined 1052 Bogalusa Heart Study participants (34.98% African American, 57.41% female, aged 33.6-57.5 years). Measures of LV mass and relative wall thickness (RWT) were obtained using two-dimensional-guided echocardiographic measurements via validated eqs. LV mass was indexed to height2.7 to calculate left ventricular mass index (LVMI). Untargeted metabolomic analysis of fasting serum samples was conducted. In combined and ethnicity-stratified analyses, multivariable linear and multinomial logistic regression models tested the associations of metabolites with the continuous LVMI and RWT and categorical LV geometry phenotypes, respectively, after adjusting for demographic and traditional cardiovascular disease risk factors. RESULTS: Pseudouridine (B = 1.38; p = 3.20 × 10-5) and N-formylmethionine (B = 1.65; 3.30 × 10-6) were significantly associated with LVMI in the overall sample as well significant in Caucasians, with consistent effect direction and nominal significance (p < .05) in African Americans. Upon exclusion of individuals with self-report myocardial infarction or congestive HF, we similarly observed a 1.33 g/m2.7 and 1.52 g/m2.7 higher LVMI for each standard deviation increase in pseudouridine and N-formylmethionine, respectively. No significant associations were observed for metabolites with RWT or categorical LV remodeling outcomes. CONCLUSIONS: The current analysis identified novel associations of pseudouridine and N-formylmethionine with LVMI, suggesting that mitochondrial-derived metabolites may serve as early biomarkers for LV remodeling and subclinical HF.

Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.

Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.

Poly-MVA attenuates 7,12- dimethylbenz[a]anthracene initiated and croton oil promoted skin papilloma formation on mice skin.

OBJECTIVE: Chemopreventive agents which exhibit activities such as anti-inflammation, inhibition of carcinogen induced mutagenesis and scavenging of free radical might play a decisive role in the inhibition of chemical carcinogenesis either at the initiation or promotion stage. Many synthesized palladium (Pd) complexes tested experimentally for antitumor activity are found effective. Poly-MVA is a liquid blend preparation containing B complex vitamins, ruthenium with Pd complexed with alpha lipoic acid as the major ingredients. The antitumor effect of Poly-MVA was evaluated against 7,12-dimethylbenz[a] anthracene-initiated croton oil-promoted papilloma formation on mice skin. Skin tumor was initiated with a single application of 390 nmol of DMBA in 20 µl acetone. The effect of Poly-MVA against croton oil- induced inflammation and lipid peroxidation on the mice skin was also evaluated. Topical application of Poly-MVA (100 µl, twice weekly for 18 weeks) 30 minutes prior to each croton oil application, significantly decreased the tumor incidence (11%) and the average number of tumor per animals. Application of Poly-MVA (100 µl) before croton oil significantly (p &#60; 0.05) protected the mouse skin from inflammation (36%) and lipid peroxidation (14%) when compared to the croton oil alone treated group. Experimental results indicate that Poly-MVA attenuate the tumor promoting effects of croton oil and the effect may probably be due to its anti-inflammatory and antioxidant activity.

A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing.

Messenger-RNA-directed protein synthesis is accomplished by the ribosome. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNA(fMet)). The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF); it removes this formyl group as polypeptides emerge from the ribosomal tunnel and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal alpha-helical extension. Studies focusing on PDF as a target for antibacterial drugs have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 A resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.

Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus.

BACKGROUND: Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. RESULTS: In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. CONCLUSIONS: These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

IMPORTANCE: Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19.

Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Deformylation of nascent peptide chains on the ribosome.

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.

Mitochondrial DNA variants modulate N-formylmethionine, proteostasis and risk of late-onset human diseases.

Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis.

Novel structural determinants of the human neutrophil chemotactic activity of leukotriene B.

A specific 5(S),12(R)-dihydroxy-eicosa-6,8,10(trans/trans/cis), 14(cis)-tetraenoic acid, designated leukotriene B, is generated by the lipoxygenation and subsequent enzymatic hydration of arachidonic acid in a variety of leukocytes. Leukotriene B elicits a maximal human neutrophil chemotactic response in vitro which is similar in magnitude to those evoked by the chemotactic fragment of the fifth component of complement, C5a, synthetic formyl-methionyl peptides, and 5-hydroxy-eicosatetraenoic acid (5-HETE). The neutrophil chemotactic potency of purified leukotriene B, assessed by the 50% effective concentration of 6 x 10(-9) M, is equivalent to that of C5a, but is up to 100-fold greater than that of 5-HETE and of other natural di-HETE isomers. 5(S),12(R)-di-hydroxy-eicosa-6,8,10(all-trans),14(cis)-tetraenoic acid, which differs from leukotriene B only in having a trans-double bond in place of a cis-double bond in the triene portion of the molecule, and acetyl-leukotriene B are significantly less potent neutrophil chemotactic factors than leukotriene B, which indicates that both the conjugated double bonds and the free hydroxyl-group(s) are functionally critical determinants. The capacity of acetyl-leukotriene B to inhibit competitively and selectively the human neutrophil chemotactic response to equimolar concentrations of leukotriene B suggests the existence of a specific subset of receptors for this potent lipid mediator.

Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes.

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10-fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.

Chemiluminescence of phagocytic cells caused by N-formylmethionyl peptides.

N-formylmethionyl (F-Met) peptides, when added alone to macrophages or polymorphonuclear leukocytes (PMN), were found to induce a chemiluminescent response of shorter duration than that produced by the commonly employed particulate stimulant, zymosan. The cellular nature of F-Met peptide-induced chemiluminescence was indicated by its dependence on cell concentration, and by its inhibition by cell disruption, heat inactivation, or previous maximal stimulation by the peptides. Comparison of PMN and macrophages from different species showed that the maximal chemiluminescent response seen in the dose-response curve of F-Met- Phe was different in different cell types. Chemiluminescence reached highest values in human PMN, it was intermediate in guinea pig macrophages and PMN, and in rabbit PMN; but it was nonexistent in rabbit alveolar macrophages and very low in rabbit peritoneal macrophages. A definite relationship was observed between peptide structure and chemiluminescent activity. Met-Phe, F- Met and Phe were inactive even at millimolar concentrations, while F-Met-Phe caused chemiluminescence at micromolar concentrations. Four active peptides were tested in guinea pig, rabbit, and human PMN, and in guinea pig alveolar and peritoneal macrophages. The relative activity of these peptides was the same in all cells studied, e.g. F-Met-Leu-Phe >> F-Met-Phe > F-Met-Val > F- Met-Ala. The values of ED50 for each peptide were also comparable to previously reported ED50 values of these peptides in inducing lysosomal enzyme release. These results were seen both in the presence and absence ofthe chemiluminescent oxidant indicator, luminol. Low concentrations of superoxide dismutase (10 mug/ml) completely inhibited chemiluminescence caused by the F-Met peptides, suggesting the involvement of 0(2)(-) or O(2)(-)-derived compounds in this response. Sodium azide, an inhibitor of peroxidase reactions, had either no effect or a slight inhibitory effect on chemiluminescence. However, when the extracellular release of lysosomal enzymes was induced by cytochalasin B, an azide- inhibitable enhancement of chemiluminescence was seen in PMN, but not in macrophages. This effect appears to be correlated with the presence of granule-associated myeloperoxidase. Although azide-inhibitable peroxidases could be a potential source of light, they did not appear to be a significant contributor in these experiments. Based on these results and on those of previous investigators, we postulate that the F-Met-peptides stimulate 0(2)(-) production in addition to stimulating lysosomal enzyme release and chemotaxis. The similar structure- activity relationship which appears to exist for these processes may indicate that they are all initiated by a single receptor mechanism. Since F-Met peptides are formed in bacteria it is likely that their actions represent an important physiologic response.

Development of specific receptors for N-formylated chemotactic peptides in a human monocyte cell line stimulated with lymphokines.

A human monocyte-like cell line, U937, when grown in continuous culture, does not secrete lysosomal enzymes or migrate towards chemotactic factors. When the cells are stimulated by lymphokines, however, they develop the ability both to migrate directionally and to secrete enzymes in response to several types of chemoattractants. The development, by stimulated cells, of chemotactic and secretory responses to one class of chemoattractants, the N- formylated peptides, is accompanied by the appearance on the cells of specific binding sites for these substances. Using tritiated N-formyl- methionyl-leueyl-phenylalanine (fMet-Leu-[(3)H]Phe) as a ligand, it was determined that unstimulated U937 cells possess no detectable binding sites. However, after stimulation with lymphocyte culture supernates for 24, 48, and 72 h, they developed 4,505 (+/-) 1,138, 22,150(+/-) 4,030, and 37,200 (+/-) 8,000 sites/cell, respectively. The dissociation constants for the interaction of fMet-Leu-[SH]Phe with the binding sites were approximately the same regardless of stimulation time and ranged between 15 and 30 nM. The binding of fMet-Leu-[(3)H]Phe by stimulated U937 cells was rapid and readily reversed by the addition of a large excess of unlabeled peptide. The affinity of a series of N-formylated peptides for binding to U937 cells exactly reflected the potency of the peptides in inducing lysosomal enzyme secretion and chemotaxis. The availability of a continuous human monocytic cell line that can be induced to express receptors for N-formylated peptides will provide a useful tool not only for the characterization of such receptors but also for the delineation of regulatory mechanisms involved in cellular differentiation and the chemotactic response.

The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils.

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.

Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis.

Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb). However, only a few Mtb peptides recognized by MHC class Ia-restricted CD8(+) T cells have been identified. Information on epitopes recognized by class Ib-restricted T cells is even more limited. M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8(+) T cells. Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes. We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides. Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay. Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. The Mtb peptide-specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells. Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner. Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.

Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils.

Mitochondria synthesize several hydrophobic proteins. Like bacteria, mitochondria initiate protein synthesis with an N-formylmethionine residue. Because N-formylmethionyl peptides have been found to be chemotactic for polymorphonuclear leukocytes (PMN), mitochondria isolated from cultured human cells and purified bovine mitochondrial proteins were tested for PMN chemotactic activity in vitro. Nondisrupted mitochondria were not chemotactic. However, intact mitochondria that had been incubated with a lysosomal lysate did stimulate PMN migration. Antibodies directed against two mitochondrial enzymes, cytochrome oxidase and ATPase, (both of which contain mitochondrially synthesized subunits) but not anti-C3 or anti-C5 decreased mitochondrially derived chemotactic activity. In addition, purified bovine mitochondrial N-formylmethionyl proteins stimulated PMN migration in vitro, whereas nonformylated mitochondrial proteins did not. Furthermore, the chemotactic activity of purified mitochondrial proteins and disrupted mitochondria was decreased by the formyl peptide antagonist butyloxycarbonyl-phenylalanine-leucine-phenylalanine-leucine-phenylalanine. Finally, disrupted mitochondria and purified mitochondrial proteins stimulated PMN-directed migration (chemotaxis), according to accepted criteria. In addition to other chemotactic factors, release of N-formylmethionyl proteins from mitochondria at sites of tissue damage, may play a role in the accumulation of inflammatory cells at these sites.

The majority of H2-M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides.

We used a new monoclonal antibody (mAb 130) to analyze the intracellular trafficking and surface expression of H2-M3, the major histocompatibility complex class Ib molecule that presents N-formylated peptides to cytotoxic T cells. M3 surface expression is undetectable in most cell types due to the paucity of endogenous antigen. M3 is induced on the cell surface by addition of high-affinity N-formylated peptides from mitochondria and listeria. Peptide-induced M3 expression is most efficient on antigen presenting cells. Basal and inducible expression of M3 is transporter associated with antigen processing (TAP)-dependent, distinguishing M3 from the class Ib molecules TL and CD1. Unlike the expression of class Ia molecules and a previously described M3/L(d) chimera, surface expression of M3 cannot be rescued by lowered temperature, suggesting that the alpha3 domain and transmembrane region of M3 may control trafficking. Pulse-chase analysis and use of trafficking inhibitors revealed a pool of empty M3 in the endoplasmic reticulum or early Golgi apparatus. Addition of exogenous peptide allows maturation with kinetics matching those of D(d). The lack of endogenous N-formylated peptide allows discovery of novel pathogen-derived peptides in normal antigen presenting cells. The nonpolymorphic nature of M3 and its ability to present bacterial antigens rapidly and dominantly make it an attractive target for peptide vaccination strategies.