Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

Expression and function of spineless orthologs correlate with distal deutocerebral appendage morphology across Arthropoda.

The deutocerebral (second) head segment is putatively homologous across Arthropoda, in spite of remarkable disparity of form and function of deutocerebral appendages. In Mandibulata this segment bears a pair of sensory antennae, whereas in Chelicerata the same segment bears a pair of feeding appendages called chelicerae. Part of the evidence for the homology of deutocerebral appendages is the conserved function of homothorax (hth), which has been shown to specify antennal or cheliceral fate in the absence of Hox signaling, in both mandibulate and chelicerate exemplars. However, the genetic basis for the morphological disparity of antenna and chelicera is not understood. To test whether downstream targets of hth have diverged in a lineage-specific manner, we examined the evolution of the function and expression of spineless (ss), which in two holometabolous insects is known to act as a hth target and distal antennal determinant. Toward expanding phylogenetic representation of gene expression data, here we show that strong expression of ss is observed in developing antennae of a hemimetabolous insect, a centipede, and an amphipod crustacean. By contrast, ss orthologs are not expressed throughout the cheliceral limb buds of spiders or harvestmen during developmental stages when appendage fate is specified. RNA interference-mediated knockdown of ss in Oncopeltus fasciatus, which bears a simple plesiomorphic antenna, resulted in homeotic distal antenna-to-leg transformation, comparable to data from holometabolous insect counterparts. Knockdown of hth in Oncopeltus fasciatus abrogated ss expression, suggesting conservation of upstream regulation. These data suggest that ss may be a flagellar (distal antennal) determinant more broadly, and that this function was acquired at the base of Mandibulata.

A Comparative Evaluation of Commercially Available Cell-Based Allografts in a Rat Spinal Fusion Model.

BACKGROUND: To evaluate the comparative abilities of commercially available, viable, cellular bone allografts to promote posterolateral spinal fusion. METHODS: Human allografts containing live cells were implanted in the athymic rat model of posterolateral spine fusion. Three commercially available allogeneic cellular bone matrices (Trinity Evolution, Trinity ELITE and Osteocel Plus) were compared with syngeneic iliac crest bone as the control. All spines underwent radiographs, manual palpation, and micro-computed tomography (CT) analysis after excision at 6 weeks. Histological sections of randomly selected spines were subjected to semiquantitative histopathological scoring for bone formation. RESULTS: By manual palpation, posterolateral fusion was detected in 40% (6/15) of spines implanted with syngeneic bone, whereas spines implanted with Trinity Evolution and Trinity ELITE allografts yielded 71% (10/14) and 77% (10/13) fusion, respectively. Only 7% (1/14) of spines implanted with Osteocel Plus allografts were judged fused by manual palpation (statistically significantly less than ELITE, P < .0007, and Evolution, P < .0013). The mineralized cancellous bone component of the allografts confounded radiographic analysis, but Trinity Evolution (0.452 ± 0.064) and Trinity ELITE (0.536 ± 0.109) allografts produced statistically significantly higher bone fusion mass volumes measured by quantitative micro-CT than did syngeneic bone (0.292 ± 0.109, P < .0001 for ELITE and P < .003 for Evolution) and Osteocel Plus (0.258 ± 0.103, P < .0001). Semiquantitative histopathological scores supported these findings because the total bone and bone marrow scores reflected significantly better new bone and marrow formation in the Trinity groups than in the Osteocel Plus group. CONCLUSIONS: The Trinity Evolution and Trinity ELITE cellular bone allografts were more effective at creating posterolateral fusion than either the Osteocel Plus allografts or syngeneic bone in this animal model. CLINICAL RELEVANCE: The superior fusion rate of Trinity cellular bone allografts may lead to better clinical outcome of spinal fusion surgeries.

Hypoxia-inducible factor 3-alpha expression is associated with the stable chondrocyte phenotype.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes.

INTRODUCTION: Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation. METHODS: Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured. RESULTS: In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time. CONCLUSIONS: Hypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.