Özlem Önder

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.

OspC Is Potent Plasminogen Receptor on Surface of Borrelia burgdorferi

Background: Binding of the vertebrate protease plasminogen is critical during the infectious cycle of Borrelia burgdorferi, although the mechanism of immobilization is unknown. Results: Only OspC-expressing spirochetes immobilize plasminogen. Conclusion: OspC, a dominant surface protein during the tick-to-host transition, is a potent plasminogen receptor. Significance: Determining the timing and location of specific protein partnerships is crucial to understanding the infectious cycle. Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.

N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

The cytochrome c maturation components CcmF, CcmH and CcmI form a membrane-integral multisubunit heme ligation complex

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

Identifying sources of tick blood meals using unidentified tandem mass spectral libraries

Rapid and reliable identification of the vertebrate species on which a disease vector previously parasitized is imperative to study ecological factors that affect pathogen distribution and can aid the development of public health programs. Here we describe a proteome profiling technique designed to identify the source of blood meals of hematophagous arthropods. This method employs direct spectral matching and thus does not require a priori knowledge of any genetic or protein sequence information. Using this technology, we detect remnants of blood in blacklegged ticks (Ixodes scapularis) and correctly determine the vertebrate species from which the blood was derived even six months after the tick had fed. This biological fingerprinting methodology is sensitive, fast, cost-effective, and can potentially be adapted for other biological and medical applications when existing genome-based methods are impractical or ineffective.

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.

Overproduction or Absence of the Periplasmic Protease DegP Severely Compromises Bacterial Growth in the Absence of the Dithiol: Disulfide Oxidoreductase DsbA

Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in photosynthesis but are defective in respiration especially in enriched growth medium at 35 °C. They also exhibit severe pleiotropic phenotypes extending from motility defects to osmofragility and oxidative stresses. In this work, using a combined proteomics and molecular genetics approach, we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originates from the overproduction of the periplasmic protease DegP, which renders them temperature-sensitive for growth. The DsbA-null mutants reverted frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. In agreement with these findings, we showed that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for its protease-chaperone activity. Remarkably although R. capsulatus DsbA-null or DegP-null mutants were viable, DegP-null DsbA-null double mutants were lethal at all growth temperatures. This is unlike Escherichia coli, and it indicates that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations among the DegP homologues, including a critical disulfide bond-bearing region, correlates well with the differences seen between various species like R. capsulatus and E. coli. Our findings illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.

Modifications of the Lipoamide-containing Mitochondrial Subproteome in a Yeast Mutant Defective in Cysteine Desulfurase

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.

Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fundamental role in understanding cell development, heritable characters and progression of diseases. Histone post-translational modifications (PTMs) are major regulators of the epigenetic machinery due to their ability to modulate gene expression, DNA repair and chromosome condensation. Large-scale strategies based on mass spectrometry have been impressively improved in the last decade, so that global changes of histone PTM abundances are quantifiable with nearly routine proteomics analyses and it is now possible to determine combinatorial patterns of modifications. Presented here is an overview of the most utilized and newly developed proteomics strategies for histone PTM characterization and a number of case studies where epigenetic mechanisms have been comprehensively characterized. Moreover, a number of current epigenetic therapies are illustrated, with an emphasis on cancer.

Reductions in Human Lyme Disease Risk Due to the Effects of Oral Vaccination on Tick-to-Mouse and Mouse-to-Tick Transmission

Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks from pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. To successfully limit human disease risk, vaccines targeting the wildlife reservoirs of B. burgdorferi must be easily distributable and must effectively reduce pathogen transmission from infected animals, given that many animals in nature will be infected prior to vaccination. We assessed the efficacy of an easily distributable oral bait vaccine based on the immunogenic outer surface protein A (OspA) to protect uninfected mice from infection and to reduce transmission from previously infected white-footed mice, an important reservoir host of B. burgdorferi. Oral vaccination of white-footed mice effectively reduces transmission of B. burgdorferi at both critical stages of the Lyme disease transmission cycle. First, oral vaccination of uninfected white-footed mice elicits an immune response that protects mice from B. burgdorferi infection. Second, oral vaccination of previously infected mice significantly reduces the transmission of B. burgdorferi to feeding ticks despite a statistically nonsignificant immune response. We used the estimates of pathogen transmission to and from vaccinated and unvaccinated mice to model the efficacy of an oral vaccination campaign targeting wild white-footed mice. Projection models suggest that the effects of the vaccine on both critical stages of the transmission cycle of B. burgdorferi act synergistically in a positive feedback loop to reduce the nymphal infection prevalence, and thus human Lyme disease risk, well below what would be expected from either effect alone. This study suggests that oral immunization of wildlife with an OspA-based vaccine can be a promising long-term strategy to reduce human Lyme disease risk.