LRPPRC and SLIRP synergize to maintain sufficient and orderly mammalian mitochondrial translation.

In mammals, the leucine-rich pentatricopeptide repeat protein (LRPPRC) and the stem-loop interacting RNA-binding protein (SLIRP) form a complex in the mitochondrial matrix that is required throughout the life cycle of most mitochondrial mRNAs. Although pathogenic mutations in the LRPPRC and SLIRP genes cause devastating human mitochondrial diseases, the in vivo function of the corresponding proteins is incompletely understood. We show here that loss of SLIRP in mice causes a decrease of complex I levels whereas other OXPHOS complexes are unaffected. We generated knock-in mice to study the in vivo interdependency of SLIRP and LRPPRC by mutating specific amino acids necessary for protein complex formation. When protein complex formation is disrupted, LRPPRC is partially degraded and SLIRP disappears. Livers from Lrpprc knock-in mice had impaired mitochondrial translation except for a marked increase in the synthesis of ATP8. Furthermore, the introduction of a heteroplasmic pathogenic mtDNA mutation (m.C5024T of the tRNAAla gene) into Slirp knockout mice causes an additive effect on mitochondrial translation leading to embryonic lethality and reduced growth of mouse embryonic fibroblasts. To summarize, we report that the LRPPRC/SLIRP protein complex is critical for maintaining normal complex I levels and that it also coordinates mitochondrial translation in a tissue-specific manner.

Mitochondria and Hypoxia: Metabolic Crosstalk in Cell-Fate Decisions.

Alterations in mitochondrial metabolism influence cell differentiation and growth. This process is regulated by the activity of 2-oxoglutarate (2OG)-dependent dioxygenases (2OGDDs) - a diverse superfamily of oxygen-consuming enzymes - through modulation of the epigenetic landscape and transcriptional responses. Recent reports have described the role of mitochondrial metabolites in directing 2OGDD-driven cell-fate switches in stem cells (SCs), immune cells, and cancer cells. An understanding of the metabolic mechanisms underlying 2OGDD autoregulation is required for therapeutic targeting of this system. We propose a model dependent on oxygen and metabolite availability and discuss how this integrates 2OGDD metabolic signalling, the hypoxic transcriptional response, and fate-determining epigenetic changes.

The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

Mitochondrial Protein Lipoylation and the 2-Oxoglutarate Dehydrogenase Complex Controls HIF1α Stability in Aerobic Conditions.

Hypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

A Comparison of Short-Term Outcomes of Minimally Invasive Computer-Assisted vs Minimally Invasive Conventional Instrumentation for Primary Total Knee Arthroplasty: A Systematic Review and Meta-Analysis.

BACKGROUND: Median parapatellar approach is the most used for total knee arthroplasty (TKA). With the advent of enhanced recovery and shorter length of hospital stay, there is an increasing pressure on surgeons to perform surgery through smaller incisions. Minimally invasive (MIS) TKA allows earlier functional recovery; it is not clear if this is associated with more complications. It is also unclear if computer-assisted minimally invasive (MIS CA) TKA has any affect on improving patient outcomes. We performed a systematic review and meta-analysis comparing MIS CA vs MIS TKA. METHODS: We performed an extensive literature search including both randomized controlled studies and prospective cohort studies. All data reported on component alignment, surgical time, complications, knee flexion, and postoperative functional knee scores were included for analysis. RESULTS: Ten studies were suitable for inclusion resulting in 490 patients with MIS CA and 503 MIS patients. There was no significant difference in the outliers on complications, knee flexion, and postoperative functional scores. Coronal plane tibial component showed statistically significant number of outliers in the MIS group demonstrating superior component positioning in the MIS CA group. Operative time was significantly longer in the MIS CA group with a mean increase of 32 minutes. CONCLUSIONS: Computer-assisted minimally invasive TKA is superior than the standard MIS TKA in terms of component positioning; however, it is unclear if this will have any long-term clinical implications. The increased operative time, although clinically relevant, does not appear to be associated with an increase in complications.

Mesenchymal stromal cells and regulatory T cells: the Yin and Yang of peripheral tolerance?

In recent years, mesenchymal stromal cells (MSCs) and regulatory T cells (Tregs) have both garnered significant interest from immunologists worldwide, not least because of the potential application of both cell types in the treatment of many chronic inflammatory and autoimmune diseases. Although both MSCs and Tregs can be considered immunosuppressive in their own right, the induction of Tregs by activated MSCs is now a well-publicised phenomenon; however, only recently have the mechanisms involved in this induction started to become clear. Indeed, it is becoming increasingly apparent that there exists a complex interplay between the two lineages leading to this potent inhibition of the host immune response. Cell contact, soluble mediators-including prostaglandin E(2) and transforming growth factor ß-and indirect induction via manipulation of other antigen-presenting cells all appear to have vital roles in the interactions between MSCs and Tregs. Much still remains to be discovered before we have a full understanding of this important aspect of the immune response, but there have already been a multitude of clinical trials suggesting that MSC/Treg therapies could offer significant benefits in the treatment of both autoimmune disease and graft versus host disease. Although these therapies are still in their infancy, the synergy between MSCs and Tregs will undoubtedly yield future breakthroughs in the treatment of many debilitating conditions and usher in a new wave of targeted, cell-based therapeutics.

Potential evidence for biotype-specific chemokine profile following BVDV infection of bovine macrophages.

Chemokines play a key role in initiating the innate and subsequently adaptive immune response by recruiting immune cells to the site of an infection. Monocytes/macrophages (MØ) are part of the first line of defence against invading pathogens, and have been shown to release a variety of chemokines in response to infection. Here, we reveal the early transcriptional response of MØ to infection with cytopathogenic (cp) and non-cytopathogenic (ncp) bovine viral diarrhoea strains (BVDV). We demonstrate up-regulation of several key chemokines of the CCL and CXCL families in MØ exposed to cpBVDV, but not ncpBVDV. In contrast, infection of MØ with ncpBVDV led to down-regulation of chemokine mRNA expression compared to uninfected cells. Data suggest that ncpBVDV can shut down production of several key chemokines that play crucial roles in the immune response to infection. This study helps to further our understanding of the pathogenesis of BVDV infection, highlighting biotype-specific cellular responses.