Unusual Post-translational Modification Research Articles

EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data.

Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family .

Molecular and Physiological Characterization of a Receptor for d-Amino Acid-Containing Neuropeptides.

Neuropeptides in several animals undergo an unusual post-translational modification, the isomerization of an amino acid residue from the l-stereoisomer to the d-stereoisomer. The resulting d-amino acid-containing peptide (DAACP) often displays biological activity higher than that of its all-l-residue analogue, with the d-residue being critical for function in many cases. However, little is known about the full physiological roles played by DAACPs, and few studies have examined the interaction of DAACPs with their cognate receptors. Here, we characterized the signaling of several DAACPs derived from a single neuropeptide prohormone, the Aplysia californica achatin-like neuropeptide precursor (apALNP), at their putative receptor, the achatin-like neuropeptide receptor (apALNR). We first used quantitative polymerase chain reaction and in situ hybridization experiments to demonstrate receptor (apALNR) expression throughout the central nervous system; on the basis of the expression pattern, we identified novel physiological functions that may be mediated by apALNR. To gain insight into ligand signaling through apALNR, we created a library of native and non-native neuropeptide analogues derived from apALNP (the neuropeptide prohormone) and evaluated them for activity in cells co-transfected with apALNR and the promiscuous Gα subunit Gα-16. Several of these neuropeptide analogues were also evaluated for their ability to induce circuit activity in a well-defined neural network associated with feeding behavior in intact ganglia from Aplysia. Our results reveal the specificity of apALNR and provide strong evidence that this receptor mediates diverse physiological functions throughout the central nervous system. Finally, we show that some native apALNP-derived DAACPs exhibit enhanced stability toward endogenous proteases, suggesting that the d-residues in these DAACPs may increase the peptide lifetime, in addition to influencing receptor specificity, in the nervous system. Ultimately, these studies provide insight into signaling at one of the few known DAACP-specific receptors and advance our understanding of the roles that l- to d-residue isomerization play in neuropeptide signaling.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea.

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.

Identification of short single disulfide-containing contryphans from the venom of cone snails using de novo mass spectrometry-based sequencing methods

We identified 12 short single disulfide-containing conopeptides from the venom of Conus coronatus, C. leopardus, C. lividus and C. zonatus. Interestingly, we detected the shortest contryphan sequence thus far characterized which contains only six amino acid residues. We also identified three distinct contryphan sequences of C. lividus without any proline residues and one sequence with an unusual post-translational modification (bromination of tryptophan). Furthermore, we characterized venom peptides of C. zonatus for the first time.

Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips

Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Because of its metal-chelating property, the histidine residue is easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesized that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >4200 proteins. Ten interacting proteins were identified, and successfully validated using a third peptide probe, fluorescence polarization assays, as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed that our interacting proteins were enriched in oxido-reduction processes, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. Besides, we found that the consensus binding motif among our identified proteins, including bacteria and human, were located within α-helices and faced the outside of proteins. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, and sensitive enough to detect even the insertion of a single oxygen atom in this case.

Top-Down Mass Spectrometry Analysis of Membrane-Bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides.

We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 β polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the β polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid.

Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A.

Synthesis of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity, thus allowing translation to resume. In their active form, both EF-P and e/aIF-5A are post-translationally modified at a positively charged residue, which protrudes toward the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual posttranslational protein modifications.

Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor.

Pupylation is a posttranslational modification peculiar to actinobacteria wherein proteins are covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated protein degradation in mycobacteria. Here, we report studies of pupylation in a streptomycete that is phylogentically related to mycobacteria. We constructed mutants of Streptomyces coelicolor lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these mutants share a high susceptibility to oxidative stress compared to that of the wild-type strain. Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking genes encoding the 20S proteasome. Our findings shed new light on this unusual posttranslational modification and its role in Streptomyces physiology. The presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have motivated reevaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome system has been studied extensively in mycobacteria, it is much less understood in streptomycetes, members of a large genus of actinobacteria known for highly choreographed life cycles in which phases of morphological differentiation, sporulation, and secondary metabolism are often regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces coelicolor for the first time and present new evidence that links pupylation and the oxidative stress response in this bacterium. Surprisingly, we found that the Pup ligase has a Pup-independent role in sporulation.

Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink.

Streptococcal bacteria use peptide signals as a means of intraspecies communication. These peptides can contain unusual post-translational modifications providing opportunities for expanding our understanding of Nature's chemical and biosynthetic repertoires. Herein we have combined tools from natural products discovery and mechanistic enzymology to report the structure and biosynthesis of streptide, a streptococcal macrocyclic peptide. We show that streptide bears an unprecedented post-translational modification involving a covalent linkage between two unactivated carbons within the side chains of lysine and tryptophan. The biosynthesis of streptide was addressed by genetic and biochemical studies. The former implicated a new SPASM domain-containing radical SAM enzyme, StrB, while the latter revealed that StrB contains two [4Fe-4S] clusters and installs the unusual lysine-to-tryptophan crosslink in a single step. By intramolecularly stitching together the side chains of lysine and tryptophan, StrB provides a new route for biosynthesizing macrocyclic peptides.

The Cys-Tyr cross-link of cysteine dioxygenase changes the optimal pH of the reaction without a structural change.

Cysteine dioxygenase (CDO) is a non-heme monoiron enzyme with an unusual posttranslational modification in the proximity of the ferrous iron active site. This modification, a cysteine to tyrosine thioether bond, cross-links two β-strands of the β-barrel. We have investigated its role in catalysis through a combined crystallographic and kinetic approach. The C93G variant lacks the cross-link and shows little change in structure from that of the wild type, suggesting that the cross-link does not stabilize an otherwise unfavorable conformation. A pH-dependent kinetic study shows that both cross-linked and un-cross-linked CDO are active but the optimal pH decreases with the presence of the cross-link. This result reflects the effect of the thioether bond on the pKa of Y157 and this residue's role in catalysis. At higher pH values, kcat is also higher for the cross-linked form, extending the pH range of activity. We therefore propose that the cross-link also increases activity by controlling deleterious interactions involving the thiol/ate of C93.

Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly.

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein production.

Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly (LB185)

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine‐243, a residue that contacts the 25S rRNA near the P‐site. Rpl3p methylation is dependent upon Hpm1p, a candidate seven beta strand methyltransferase. In this study, we characterized Hpm1p activity in vitro and in vivo. Amino acid analysis of cell lysates and subcellular fractions reveals that Hpm1p is responsible for all detectable histidine protein methylation. The modification is found in a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nuclei‐containing organelle fraction, but was not detected in proteins of the ribosome‐free cytosol fraction. In vitro assays demonstrate that Hpm1p is a methyltransferase that catalyzes the modification of ribosome‐associated but not free Rpl3p, suggesting that its activity depends on interactions of Rpl3p with other ribosomal components. hpm1 null mutant cells are defective in early rRNA processing resulting in a deficiency of 60S subunits and decreased translational activity in minimal media. Finally, cells lacking Hpm1p are resistant to cycloheximide, indicative of ribosome structure abnormalities. We propose that Hpm1p plays a role in orchestrating the assembly of biogenesis factors during early ribosome assembly.Grant Funding Source: NIH

Unusual post-translational protein modifications: the benefits of sophistication

This review summarizes the “seemingly bizarre”, yet naturally occurring, covalent non-disulphide cross-links in enzymatic and scaffolding proteins and their functions.

Class D β-Lactamases: A Reappraisal after Five Decades

Despite 70 years of clinical use, β-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., β-lactamases) that can hydrolyze the β-lactam bond and inactivate the antibiotic. These enzymes can be grouped into four classes (A-D). Among the most genetically diverse are the class D β-lactamases. In this class are β-lactamases that can inactivate the entire spectrum of β-lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D β-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa , Escherichia coli , Proteus mirabilis , and Acinetobacter baumannii . The active-sites of class D β-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D β-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum β-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (e.g., cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D β-lactamases that hydrolyze clinically important carbapenem β-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow-spectrum β-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D β-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of β-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both β-lactam derivatives and non-β-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a β-lactam antibiotic, and others show a unique mechanism of inhibition that is distinct from the traditional β-lactamase inhibitors. These studies will bolster structure-based inhibitor design efforts to facilitate the optimization and development of these compounds as class D inactivators.

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1', and P2' positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1' position. The P2' position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2' Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.

Defining Intact Protein Primary Structures from Saliva: A Step toward the Human Proteome Project

Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of "unique" ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.

Posttranslational β-methylation and macrolactamidination in the biosynthesis of the bottromycin complex of ribosomal peptide antibiotics

Bottromycins are unique peptide antibiotics that contain a macrolactamidine, several non-proteinogenic amino acids and a thiazole. We hypothesised that the bottromycins originate from a ribosomally biosynthesised precursor that is elaborated to the mature antibiotics via a series of unusual posttranslational modifications, including β-methylation of Phe, Val and Pro residues, and proteolytic macrolactamidine formation. To investigate this hypothesis, we generated a draft genome sequence of the bottromycin producer Streptomyces bottropensis DSM 40262 in which we identified a gene encoding a polypeptide containing a sequence corresponding to the putative precursor of the bottromycins. Deletion of the gene encoding this putative precursor peptide abolished production of bottromycins in S. bottropensis. Bottromycin production was restored in the resulting mutant by in trans expression of the entire operon containing the precursor peptide gene. In contrast to other posttranslationally modified ribosomal peptide antibiotics, the bottromycin precursor peptide lacks an N-terminal “leader” sequence. Instead, it contains a C-terminal “follower” sequence that is removed during post-translational processing and that is presumably required for correct maturation of the antibiotic. Comparative sequence analyses of the proteins encoded by the genes flanking the precursor peptide gene identified ten enzymes that are likely to catalyse the posttranslational modifications required for bottromycin assembly. A gene cluster that is essentially identical to the S. bottropensis bottromycin biosynthetic gene cluster was identified in the complete genome sequence of the plant pathogen Streptomyces scabies 87.22 and LC-MS analyses confirmed that S. scabies is a novel producer of bottromycins. Our findings set the stage for engineered biosynthesis of novel bottromycin analogues and delineate a plausible pathway for bottromycin biosynthesis.

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.

RNAi knockdown of parafusin inhibits the secretory pathway

Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca 2+-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo − phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.