Faculty Bibliography

Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases.

PURPOSE: To assess the relationship between the prevalence and severity of conjunctivochalasis and pinguecula. METHODS: Cross-sectional, consecutive case study conducted at the university hospital of University of Tokyo Graduate School of Medicine. A total of 1061 patients aged from 1 to 94 years were enrolled. The grade and other parameters of conjunctivochalasis (classified into three locations: nasal, middle and temporal) and the grade of pinguecula located on the nasal or temporal conjunctiva were determined in all subjects. Patients were also divided into 5 or 10 age groups. RESULTS: The severity of conjunctivochalasis affecting the nasal and temporal bulbar conjunctiva was significantly correlated with the grade of pinguecula located on the nasal and temporal conjunctiva in each age group (p < 0.05). Pinguecula was independently associated with conjunctivochalasis (nasal: odds ratio [OR] = 1.44; temporal: OR = 1.43) after adjustment for age. CONCLUSION: This was the first assessment of the relation between the grade of conjunctivochalasis and pinguecula in a large consecutive series of patients. Our results suggest that the prevalence and severity of conjunctivochalasis are related to the presence of pinguecula.

Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin-1 and fibrillin-2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril-associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF-beta binding protein 1 (LTBP-1), -3 and -4; the three LTBPs that form a complex with TGF-beta. In Fbn1(-/-) ascending aortas and lungs, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. In addition, in cultures of Fbn1(-/-) smooth muscle cells or lung fibroblasts, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. Fibrillin-2 is not involved in the deposition of LTBP-1 in Fbn1(-/-) extracellular matrix as cells deficient for both fibrillin-1 and fibrillin-2 still incorporate LTBP-1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1(-/-) cells abrogates the deposition of LTBP-1. Together, these data indicate that LTBP-3 and LTBP-4 association with the matrix depends on fibrillin-1 microfibrils, whereas LTBP-1 association depends on a fibronectin network. J. Cell. Physiol. 227: 3828-3836, 2012. (c) 2012 Wiley Periodicals, Inc.

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1(-/-) cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1(-/-) cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

OBJECTIVES: : Recombinant human parathyroid hormone (PTH 1-34) has been previously shown to enhance fracture healing in animal models. Here, we sought to determine whether the systemic administration of PTH 1-34 is effective in preventing atrophic fracture nonunion in a murine, surgical nonunion model. METHODS: : We used an established reproducible long-bone murine fracture nonunion model by generating a midshaft femur fracture, followed by fracture distraction using an intramedullary pin and custom metallic clip to maintain a fracture gap of 1.7 mm. Mice were randomized to receive either daily intraperitoneal injections of 30 mug/kg PTH 1-34 for 14 days or saline injections. At 6 weeks after the procedure, radiographic and histologic assessment of fracture healing was performed. RESULTS: : At 6 weeks after surgery, the group treated with PTH showed higher rates of bony union (50% vs 8%; P < 0.05) as assessed by radiographic analysis. Mean gap size was also significantly lower in the PTH group (1.42 vs 0.36 mm in the control group; P < 0.05). Histologic analysis of atrophic nonunions in the control group revealed a persistent fracture gap with intervening fibrous tissue. In contrast, healed subjects in the PTH-treated group had cortical bridging with mature bone and relatively little callus, which is consistent with primary intramembranous ossification. CONCLUSIONS: : Daily systemic administration of recombinant PTH 1-34 increased the rate of union in a mouse atrophic nonunion model. This may have important implications for the potential clinical role of PTH 1-34 in the treatment of atrophic fracture nonunions.

The echinocandins caspofungin, micafungin, and anidulafungin, inhibitors of cell wall beta-1,3-glucan synthesis, were recently elevated to first-line agents for treating infections due to the azole-refractory yeast Candida glabrata. In Candida albicans, echinocandin resistance is strictly associated with mutations in Fks1, a large integral membrane protein and putative beta-1,3-glucan synthase, while mutations in both Fks1 and its paralog Fks2 (but not Fks3) have been associated with resistance in C. glabrata. To further explore their function, regulation, and role in resistance, C. glabrata fks genes were disrupted and subjected to mutational analysis, and their differential regulation was explored. An fks1Delta fks2Delta double disruptant was not able to be generated; otherwise, all three single and remaining two double disruptants displayed normal growth and echinocandin susceptibility, indicating Fks1-Fks2 redundancy. Selection on echinocandin-containing medium for resistant mutants was dependent on strain background: only fks1Delta and fks1Delta fks3Delta strains consistently yielded mutants exhibiting high-level resistance, all with Fks2 hot spot 1 mutations. Thus, Fks1-Fks2 redundancy attenuates the rate of resistance; further analysis showed that it also attenuates the impact of resistance-conferring mutations. Growth of the fks1Delta and, especially, fks1Delta fks3Delta strains was specifically susceptible to the calcineurin inhibitor FK506. Relatedly, FK506 addition or calcineurin gene CMP2 disruption specifically reversed Fks2-mediated resistance of laboratory mutants and clinical isolates. RNA analysis suggests that transcriptional control is not the sole mechanism by which calcineurin modulates Fks2 activity.

Although high high-density lipoprotein (HDL)-cholesterol levels are associated with decreased cardiovascular risk in epidemiological studies, recent genetic and pharmacological findings have raised doubts about the beneficial effects of HDL. Raising HDL levels in animal models by infusion or overexpression of apolipoprotein A-I has shown clear vascular improvements, such as delayed atherosclerotic lesion progression and accelerated lesion regression, along with increased reverse cholesterol transport. Inflammation and other factors, such as myeloperoxidase-mediated oxidation, can impair HDL production and HDL function, with regard to its reverse cholesterol transport, antioxidant, and anti-inflammatory activities. Thus, tests of HDL function, which have not yet been developed as routine diagnostic assays, may prove useful and be a better predictor of cardiovascular risk than HDL-cholesterol levels.

Rationale: Statins have been shown to have anti-inflammatory pleiotropic effects in experimental settings. However, the bioavailability of orally administered statins is poor, which limits the direct therapeutic effects on atherosclerotic plaques. We have developed a simvastatin loaded high-density lipoprotein nanoparticle platform ([S]-rHDL), with the similar properties to natural HDL (A). The formulation has potent anti-inflammatory effects in vitro (S-A), produces significantly higher therapeutic effects in apolipoprotein E knockout (ApoE KO) mice than oral simvastatin or rHDL treatment, and induces rapid regression when administered at a high dose (S-B). The purpose of the current study is to understand the mechanism of action by which this formulation exerts its potent antiinflammatory effects. Methods and Results: To investigate the protecting effects of [S]-rHDL on encapsulated simvastatin in blood, the nanoparticles were incubated in mouse serum and intact simvastatin was measured over time with HPLC. [S]-rHDL drastically protected the encapsulated simvastatin (B). To evaluate the in vivo targeting of atherosclerotic plaques, [S]-rHDL was additionally labeled with Gd-DPTA-lipids and Cy5.5-DMPE, and intravenously administered in ApoE KO mice. In vivo T1-weighted magnetic resonance imaging (MRI) revealed [S]-rHDL accumulation in atherosclerotic plaques in the abdominal aortas (C), while ex vivo nearinfrared fluorescence imaging (NIRF) of excised aortas demonstrated pronounced accumulation in plaque rich lesions such as the aortic roots and arches (D). To investigate the cellular targeting specificity of [S]-rHDL, flow cytometry and fluorescence microscopy were used to analyze the blood, spleen, and atherosclerotic plaques. [S]-rHDL was preferentially taken up by monocytes and macrophages in the blood and spleen (E), but not by other leukocytes (S-D). It also extensively accumulated in atherosclerotic plaques, and was taken up by plaque macrophages (F and S-C). Altogether these data suppor!