Ozlem Onder | University of Pennsylvania (original) (raw)

Papers by Ozlem Onder

Metallomics , 2020

Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have develop... more Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have developed sophisticated response mechanisms towards both the lack and the excess of Cu in their environments. In this study, we achieved a global view of Cu-responsive changes in the prokaryotic model organism Rhodobacter capsulatus using label-free quantitative differential proteomics. Semi-aerobically grown cells under heterotrophic conditions in minimal medium (∼0.3 μM Cu) were compared with cells supplemented with either 5 μM Cu or with 5 mM of the Cu-chelator bathocuproine sulfonate. Mass spectrometry based bottom-up proteomics of unfractionated cell lysates identified 2430 of the 3632 putative proteins encoded by the genome, producing a robust proteome dataset for R. capsulatus. Use of biological and technical replicates for each growth condition yielded high reproducibility and reliable quantification for 1926 of the identified proteins. Comparison of cells grown under Cu-excess or Cu-depleted conditions to those grown under minimal Cu-sufficient conditions revealed that 75 proteins exhibited statistically significant (p < 0.05) abundance changes, ranging from 2- to 300-fold. A subset of the highly Cu-responsive proteins was orthogonally probed using molecular genetics, validating that several of them were indeed involved in cellular Cu homeostasis.

Colony-stimulating Factor 1 Receptor (CSF1R) Activates AKT/mTOR Signaling and Promotes T-cell Lymphoma Viability, Feb 1, 2020

PURPOSE: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complet... more PURPOSE:

Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T-cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, it's aberrant expression has been reported in a subset of tumors. In this manuscript we evaluated its expression and oncogenic role in T-cell lymphomas.
EXPERIMENTAL DESIGN:

Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine-kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling.
RESULTS:

We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T-cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in-vivo and in-vitro models is associated with decreased T-cell lymphoma growth.
CONCLUSIONS:

Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb 3 -Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus, 2017

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic protei... more The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35 • C), DsbA − mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25 • C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb 3-type cytochrome c oxidase (cbb 3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu 2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb 3-Cox production in the absence of DsbA. Indeed, addition of Cu 2+ to the enriched medium suppresses, and conversely, omission of Cu 2+ from the minimal medium induces, growth and cbb 3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb 3-Cox, especially in enriched medium where bioavailable Cu 2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb 3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb 3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb 3-Cox.

Expert review of proteomics, 2015

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fun... more 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.

Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2011

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent mea... more 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 par...

Journal of Bacteriology, 2012

N-glycosylation, a posttranslational modification required for the accurate folding and stability... more 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.

Nature Communications, 2013

Rapid and reliable identification of the vertebrate species on which a disease vector previously ... more 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 haematophagous 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 6 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.

The Journal of biological chemistry, Jan 11, 2012

Host-derived proteases are crucial for the successful infection of vertebrates by several pathoge... more 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, during dissemination within the host, and 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 while only background levels of plasminogen are detectable on O...

Molecular & cellular proteomics : MCP, 2006

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulf... more 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 alpha-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 an...

Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks fro... more 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.

Nature communications, Apr 23, 2013

Rapid and reliable identification of the vertebrate species on which a disease vector previously ... more 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 haematophagous 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 6 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.

Vector borne and zoonotic diseases (Larchmont, N.Y.), 2013

Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks fro... more 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.

Journal of …, Jan 1, 2012

N-glycosylation, a posttranslational modification required for the accurate folding and stability... more 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.

Proceedings of the National Academy of Sciences of the United States of America, Jan 1, 2011

The Journal of biological chemistry, Jan 1, 2012

Background: Binding of the vertebrate protease plasminogen is critical during the infectious cycl... more 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.

Molecular & Cellular Proteomics, Jan 1, 2008

Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in pho... more 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. Molecular & Cellular Proteomics 7: 875-890, 2008.

Molecular & Cellular …, Jan 1, 2006

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulf... more 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 ␣-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.

Journal of Biological …, Jan 1, 2008

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification proces... more 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.

Metallomics , 2020

Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have develop... more Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have developed sophisticated response mechanisms towards both the lack and the excess of Cu in their environments. In this study, we achieved a global view of Cu-responsive changes in the prokaryotic model organism Rhodobacter capsulatus using label-free quantitative differential proteomics. Semi-aerobically grown cells under heterotrophic conditions in minimal medium (∼0.3 μM Cu) were compared with cells supplemented with either 5 μM Cu or with 5 mM of the Cu-chelator bathocuproine sulfonate. Mass spectrometry based bottom-up proteomics of unfractionated cell lysates identified 2430 of the 3632 putative proteins encoded by the genome, producing a robust proteome dataset for R. capsulatus. Use of biological and technical replicates for each growth condition yielded high reproducibility and reliable quantification for 1926 of the identified proteins. Comparison of cells grown under Cu-excess or Cu-depleted conditions to those grown under minimal Cu-sufficient conditions revealed that 75 proteins exhibited statistically significant (p < 0.05) abundance changes, ranging from 2- to 300-fold. A subset of the highly Cu-responsive proteins was orthogonally probed using molecular genetics, validating that several of them were indeed involved in cellular Cu homeostasis.

Colony-stimulating Factor 1 Receptor (CSF1R) Activates AKT/mTOR Signaling and Promotes T-cell Lymphoma Viability, Feb 1, 2020

PURPOSE: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complet... more PURPOSE:

Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T-cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, it's aberrant expression has been reported in a subset of tumors. In this manuscript we evaluated its expression and oncogenic role in T-cell lymphomas.
EXPERIMENTAL DESIGN:

Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine-kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling.
RESULTS:

We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T-cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in-vivo and in-vitro models is associated with decreased T-cell lymphoma growth.
CONCLUSIONS:

Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb 3 -Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus, 2017

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic protei... more The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35 • C), DsbA − mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25 • C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb 3-type cytochrome c oxidase (cbb 3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu 2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb 3-Cox production in the absence of DsbA. Indeed, addition of Cu 2+ to the enriched medium suppresses, and conversely, omission of Cu 2+ from the minimal medium induces, growth and cbb 3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb 3-Cox, especially in enriched medium where bioavailable Cu 2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb 3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb 3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb 3-Cox.

Expert review of proteomics, 2015

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fun... more 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.

Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2011

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent mea... more 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 par...

Journal of Bacteriology, 2012

N-glycosylation, a posttranslational modification required for the accurate folding and stability... more 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.

Nature Communications, 2013

Rapid and reliable identification of the vertebrate species on which a disease vector previously ... more 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 haematophagous 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 6 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.

The Journal of biological chemistry, Jan 11, 2012

Host-derived proteases are crucial for the successful infection of vertebrates by several pathoge... more 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, during dissemination within the host, and 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 while only background levels of plasminogen are detectable on O...

Molecular & cellular proteomics : MCP, 2006

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulf... more 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 alpha-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 an...

Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks fro... more 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.

Nature communications, Apr 23, 2013

Rapid and reliable identification of the vertebrate species on which a disease vector previously ... more 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 haematophagous 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 6 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.

Vector borne and zoonotic diseases (Larchmont, N.Y.), 2013

Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks fro... more 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.

Journal of …, Jan 1, 2012

N-glycosylation, a posttranslational modification required for the accurate folding and stability... more 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.

Proceedings of the National Academy of Sciences of the United States of America, Jan 1, 2011

The Journal of biological chemistry, Jan 1, 2012

Background: Binding of the vertebrate protease plasminogen is critical during the infectious cycl... more 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.

Molecular & Cellular Proteomics, Jan 1, 2008

Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in pho... more 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. Molecular & Cellular Proteomics 7: 875-890, 2008.

Molecular & Cellular …, Jan 1, 2006

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulf... more 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 ␣-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.

Journal of Biological …, Jan 1, 2008

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification proces... more 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.

Blood, 2020

ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-... more ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment.

Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function.

BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion.

To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms.

As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants.

This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients.