Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Background

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson??s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results

In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1?/? cells recapitulate the respiratory defect in isolated mitochondria from PINK1?/? mouse brains, suggesting that these PINK1?/? cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1?/? cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1?/? cells, and this genotypic difference between PINK1?/? and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1?/? cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1?/? and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1?/? compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions

Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1?/? cells.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway.Atypical hemolytic uremic syndrome (aHUS)2 is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3–8). More than half of the mutations have been found to originate in the HF1 gene that encodes FH and FH-like protein 1.The alternative pathway is initiated spontaneously by hydrolysis of C3 to C3_H2O that forms the C3-convertase C3_H2OBb (9, 10). This enzyme complex converts numerous C3 molecules to C3b that are covalently bound onto practically any nearby surface (11). On a so-called activator surface, such as a microbe, the surface-bound C3b molecules are not efficiently eliminated and therefore new C3bBb complexes are formed leading to more C3b depositions and eventually effective opsonization or damage of the target cell. On non-activator surfaces, such as viable self (host) cells, factor I cleaves C3b to inactive C3b (iC3b) in the presence of one of the cofactors (CD46, CD35, FH, and FHL-1) (12–16). FH is the only one of these cofactors that mediates recognition of self-surfaces making the alternative pathway capable of discriminating between activating and non-activating surfaces (17–19).The two main functions of FH are to prevent the alternative pathway activation in plasma and on self-surfaces. This 150-kDa glycoprotein consists of 20 tandemly arranged short consensus repeat domains that are composed of ∼60 amino acids. Domains 1–4 are essential for the cofactor and decay accelerating activity (20). In the middle region of FH (domains 5–15) there are two binding sites for C-reactive protein (21), one or two sites for glycosaminoglycans (GAGs) (22–25), and one site for C3c part of C3b (C3b/C3c) (25, 26). The C-terminal domains 19–20 (FH19–20) possess binding sites for the thiol ester domain of C3b (C3d or C3dg, TED domain) and GAGs (26, 27).The most common types of mutations found in aHUS are FH missense mutations located within FH19–20 that was recently solved as crystal and NMR structures (2, 28, 29). The C terminus of FH is crucial in self-cell protection as demonstrated by the severity of the aHUS cases and also in a recent mouse model of aHUS where domains 16–20 had been deleted (30, 31). Histopathology of aHUS in these mice had all the characteristics of human aHUS being concordant with the similarity of binding sites for C3b, heparin, and human umbilical vein endothelial cells between human and mouse FH domains 18–20 (32). Binding of mouse or human FH to glomerular endothelial cells has not been characterized despite the fact that in aHUS damage occurs mainly in the small vessels, especially in the glomeruli.The molecular pathogenesis leading to the clinical aHUS in patients with FH mutations remains elusive. The suggested molecular mechanisms for some aHUS-associated mutations include defective binding of the mutated FH to GAGs, endothelial cells, or C3b/C3d (28, 29, 33, 34). The aim of this study was to define the effects of nine aHUS-associated FH mutations and five other structurally closely located mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. We identified three primary defects of the mutants: impaired C3b/C3d binding, enhanced mGEnC-1/heparin binding, and impaired mGEnC-1/heparin binding that could lead via three mechanisms to incapability of FH to eliminate C3b on plasma-exposed self-cells. The results clarify the mechanism of target discrimination of the alternative pathway by the C terminus of FH.