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1.
J Biol Chem ; 300(5): 107236, 2024 May.
Article in English | MEDLINE | ID: mdl-38552741

ABSTRACT

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.


Subject(s)
Bacterial Outer Membrane Proteins , Borrelia burgdorferi , Complement Pathway, Classical , Humans , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Borrelia burgdorferi/immunology , Borrelia burgdorferi/metabolism , Borrelia burgdorferi/genetics , Complement C1r/metabolism , Complement C1r/genetics , Complement C1s/metabolism , Complement C1s/genetics , Complement C1s/chemistry , Complement Pathway, Classical/immunology , Lipoproteins/metabolism , Lipoproteins/genetics , Lipoproteins/chemistry , Lipoproteins/immunology , Lyme Disease/genetics , Lyme Disease/immunology , Lyme Disease/microbiology , Protein Binding
2.
J Biol Chem ; 299(8): 104972, 2023 08.
Article in English | MEDLINE | ID: mdl-37380082

ABSTRACT

Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever-causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors.


Subject(s)
Bacterial Proteins , Borrelia , Complement C1 Inactivator Proteins , Lyme Disease , Relapsing Fever , Humans , Bacterial Proteins/chemistry , Lyme Disease/immunology , Lyme Disease/microbiology , Relapsing Fever/immunology , Relapsing Fever/microbiology , Complement C1 Inactivator Proteins/chemistry , Protein Domains , Crystallography, X-Ray
3.
bioRxiv ; 2023 Mar 01.
Article in English | MEDLINE | ID: mdl-36909632

ABSTRACT

Borrelial pathogens are vector-borne etiological agents of Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind to components of the human complement system. BBK32 is an example of a borrelial lipoprotein that protects the Lyme disease spirochete from complement-mediated attack. The complement inhibitory activity of BBK32 arises from an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical pathway, C1r. Borrelia miyamotoi spirochetes encode BBK32 orthologs termed FbpA and FbpB, and these proteins also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever spirochetes, remains unknown. Here we report the crystal structure of the C-terminal domain of B. hermsii FbpC to a limiting resolution of 1.5 Å. Surface plasmon resonance studies and assays of complement function demonstrate that FbpC retains potent BBK32-like anti-complement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out 1 µs molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. This study advances our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveals a surprising plasticity in the structures of borrelial C1r inhibitors.

4.
Nat Immunol ; 24(3): 501-515, 2023 03.
Article in English | MEDLINE | ID: mdl-36797499

ABSTRACT

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.


Subject(s)
Pyrimidines , Cell Cycle , Cell Differentiation
6.
Bioconjug Chem ; 34(1): 204-211, 2023 01 18.
Article in English | MEDLINE | ID: mdl-36379001

ABSTRACT

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.


Subject(s)
Cyclic AMP-Dependent Protein Kinases , Protein Kinases , Protein Kinases/metabolism , Phosphorylation , Recombinant Fusion Proteins/chemistry , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism
7.
Front Cell Neurosci ; 16: 982074, 2022.
Article in English | MEDLINE | ID: mdl-36212686

ABSTRACT

The presence of atypical cytoskeletal dynamics, structures, and associated morphologies is a common theme uniting numerous diseases and developmental disorders. In particular, cytoskeletal dysregulation is a common cellular feature of Alzheimer's disease, Parkinson's disease, and Huntington's disease. While the numerous activators and inhibitors of dysregulation present complexities for characterizing these elements as byproducts or initiators of the disease state, it is increasingly clear that a better understanding of these anomalies is critical for advancing the state of knowledge and plan of therapeutic attack. In this review, we focus on the hallmarks of cytoskeletal dysregulation that are associated with cofilin-linked actin regulation, with a particular emphasis on the formation, monitoring, and inhibition of cofilin-actin rods. We also review actin-associated proteins other than cofilin with links to cytoskeleton-associated neurodegenerative processes, recognizing that cofilin-actin rods comprise one strand of a vast web of interactions that occur as a result of cytoskeletal dysregulation. Our aim is to present a current perspective on cytoskeletal dysregulation, connecting recent developments in our understanding with emerging strategies for biosensing and biomimicry that will help shape future directions of the field.

8.
J Mol Med (Berl) ; 100(9): 1321-1330, 2022 09.
Article in English | MEDLINE | ID: mdl-35916902

ABSTRACT

Calcification and chronic inflammation of the vascular wall is a high-risk factor for cardiovascular mortality, especially in patients with chronic uremia. For the reduction or prevention of rapid disease progression, no specific treatment options are currently available. This study aimed to evaluate an adenine-based uremic mouse model for studying medial vessel calcification and senescence-associated secretory phenotype (SASP) changes of aortic tissue to unravel molecular pathogenesis and provide a model for therapy testing. The dietary adenine administration induced a stable and similar degree of chronic uremia in DBA2/N mice with an increase of uremia blood markers such as blood urea nitrogen, calcium, creatinine, alkaline phosphatase, and parathyroid hormone. Also, renal fibrosis and crystal deposits were detected upon adenine feeding. The uremic condition is related to a moderate to severe medial vessel calcification and subsequent elastin disorganization. In addition, expression of osteogenic markers as Bmp-2 and its transcription factor Sox-9 as well as p21 as senescence marker were increased in uremic mice compared to controls. Pro-inflammatory uremic proteins such as serum amyloid A, interleukin (Il)-1ß, and Il-6 increased. This novel model of chronic uremia provides a simple method for investigation of signaling pathways in vascular inflammation and calcification and therefore offers an experimental basis for the development of potential therapeutic intervention studies.


Subject(s)
Kidney Failure, Chronic , Uremia , Vascular Calcification , Adenine/therapeutic use , Aging , Animals , Disease Models, Animal , Inflammation/complications , Mice , Rats , Rats, Sprague-Dawley , Uremia/metabolism , Uremia/pathology , Vascular Calcification/etiology
9.
Immunol Cell Biol ; 100(2): 83-86, 2022 02.
Article in English | MEDLINE | ID: mdl-34989026

ABSTRACT

A recent study by Gabriel et al. provides novel insight into the metabolic pathways that contribute to T cell differentiation in chronic infection. The researchers discovered that metabolic plasticity and the function of exhausted T cells is regulated via the TGF-ß-mTOR signaling axis.


Subject(s)
CD8-Positive T-Lymphocytes , Cell Differentiation , Lymphocyte Activation
10.
Diabetes ; 67(8): 1561-1575, 2018 08.
Article in English | MEDLINE | ID: mdl-29764859

ABSTRACT

Patients with both major forms of diabetes would benefit from therapies that increase ß-cell mass. Glucose, a natural mitogen, drives adaptive expansion of ß-cell mass by promoting ß-cell proliferation. We previously demonstrated that a carbohydrate response element-binding protein (ChREBPα) is required for glucose-stimulated ß-cell proliferation and that overexpression of ChREBPα amplifies the proliferative effect of glucose. Here we found that ChREBPα reprogrammed anabolic metabolism to promote proliferation. ChREBPα increased mitochondrial biogenesis, oxygen consumption rates, and ATP production. Proliferation augmentation by ChREBPα required the presence of ChREBPß. ChREBPα increased the expression and activity of Nrf2, initiating antioxidant and mitochondrial biogenic programs. The induction of Nrf2 was required for ChREBPα-mediated mitochondrial biogenesis and for glucose-stimulated and ChREBPα-augmented ß-cell proliferation. Overexpression of Nrf2 was sufficient to drive human ß-cell proliferation in vitro; this confirms the importance of this pathway. Our results reveal a novel pathway necessary for ß-cell proliferation that may be exploited for therapeutic ß-cell regeneration.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Gene Expression Regulation , Glucose/metabolism , Insulin-Secreting Cells/metabolism , NF-E2-Related Factor 2/agonists , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cadaver , Cell Line, Tumor , Cell Proliferation , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondrial Dynamics , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Organelle Biogenesis , Oxygen Consumption , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Interference , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Tissue Culture Techniques , Transcription Factors/chemistry , Transcription Factors/genetics
11.
Curr Biol ; 26(15): 2037-2043, 2016 08 08.
Article in English | MEDLINE | ID: mdl-27426517

ABSTRACT

Mitochondrial dysfunction is pervasive in human pathologies such as neurodegeneration, diabetes, cancer, and pathogen infections as well as during normal aging. Cells sense and respond to mitochondrial dysfunction by activating a protective transcriptional program known as the mitochondrial unfolded protein response (UPR(mt)), which includes genes that promote mitochondrial protein homeostasis and the recovery of defective organelles [1, 2]. Work in Caenorhabditis elegans has shown that the UPR(mt) is regulated by the transcription factor ATFS-1, which is regulated by organelle partitioning. Normally, ATFS-1 accumulates within mitochondria, but during respiratory chain dysfunction, high levels of reactive oxygen species (ROS), or mitochondrial protein folding stress, a percentage of ATFS-1 accumulates in the cytosol and traffics to the nucleus where it activates the UPR(mt) [2]. While similar transcriptional responses have been described in mammals [3, 4], how the UPR(mt) is regulated remains unclear. Here, we describe a mammalian transcription factor, ATF5, which is regulated similarly to ATFS-1 and induces a similar transcriptional response. ATF5 expression can rescue UPR(mt) signaling in atfs-1-deficient worms requiring the same UPR(mt) promoter element identified in C. elegans. Furthermore, mammalian cells require ATF5 to maintain mitochondrial activity during mitochondrial stress and promote organelle recovery. Combined, these data suggest that regulation of the UPR(mt) is conserved from worms to mammals.


Subject(s)
Activating Transcription Factors/genetics , Caenorhabditis elegans/genetics , Mitochondrial Proteins/genetics , Activating Transcription Factors/metabolism , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Mitochondrial Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Unfolded Protein Response
12.
Nature ; 533(7603): 416-9, 2016 05 19.
Article in English | MEDLINE | ID: mdl-27135930

ABSTRACT

Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.


Subject(s)
Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Genome, Mitochondrial/genetics , Mitochondria/genetics , Mitochondria/metabolism , Unfolded Protein Response/physiology , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , DNA, Mitochondrial/genetics , Gene Deletion , Genes, Essential/genetics , Mitochondria/pathology , Organelle Biogenesis , Oxidative Phosphorylation , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism
13.
J Cell Biol ; 211(3): 553-67, 2015 Nov 09.
Article in English | MEDLINE | ID: mdl-26553928

ABSTRACT

Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain-major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag's) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II-processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface.


Subject(s)
Actins/metabolism , Dendritic Cells/metabolism , F-Box Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Actins/immunology , Animals , Antigen Presentation/immunology , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cathepsins/metabolism , Cell Membrane/immunology , Cell Membrane/metabolism , Dendritic Cells/immunology , F-Box Proteins/immunology , F-Box-WD Repeat-Containing Protein 7 , Genes, MHC Class II/immunology , Lysosomes/immunology , Lysosomes/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ubiquitin-Protein Ligases/immunology , Up-Regulation/immunology
14.
Biochim Biophys Acta ; 1847(11): 1448-56, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25857997

ABSTRACT

Time- or age-dependent accumulation of mitochondrial damage and dysfunction is strongly associated with aging [1]. Thus, a major biomedical goal is to identify and therapeutically manipulate those inherent programs that protect against mitochondrial dysfunction to promote cell survival and organismal health. The mitochondrial unfolded protein response (UPR(mt)) is such a protective transcriptional response mediated by mitochondrial-to-nuclear signaling that includes mitochondrial proteostasis genes to stabilize mitochondrial function, metabolic adaptations, as well as an innate immunity program. Here, we review the UPR(mt) and its role during a variety of forms of mitochondrial dysfunction including those caused by mutations in respiratory chain genes as well as upon exposure to pathogens that produce mitochondrial toxins. We also review recent data in support of and against the emerging role of the UPR(mt) during aging and longevity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.


Subject(s)
Aging , Cytoprotection , Mitochondria/physiology , Unfolded Protein Response , Animals , Humans , Immunity, Innate , Phosphorylation , Protein Serine-Threonine Kinases/physiology
15.
J Chromatogr Sci ; 48(1): 55-8, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20056037

ABSTRACT

Expansion of an unstable trinucleotide (CAG)(n) repeat region within exon 1 of the gene IT15 causes autosomal, dominantly inherited Huntington's disease (HD). The number of CAG-repeats varies from 6 to 35 in normal individuals, whereas in affected patients the expanded allele contains 40 or more CAG-repeats. Thus, exact determination of both alleles of the gene (normal and expanded) on the molecular level is of great importance for clinical diagnosis and prognosis regarding the course of the disease. In our study, we optimized and evaluated a highly sensitive, automated, and economical molecular method for length characterization of the trinucleotide fragment expansion such as (CAG)(n) repeat region based on ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). We found that IP-RP-HPLC can be used for exact fragment length measuring between 60-280 bp as a sensitive and advantageous alternative method to conventional techniques.


Subject(s)
Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , DNA/analysis , Huntington Disease/diagnosis , Huntington Disease/genetics , Trinucleotide Repeat Expansion , Alleles , Chromatography, High Pressure Liquid/economics , Chromatography, Reverse-Phase/economics , DNA/genetics , Humans , Sensitivity and Specificity
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