Increased core body temperature exacerbates defective protein prenylation in mouse models of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1ß, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.

Bisphosphonate drugs have actions in the lung and inhibit the mevalonate pathway in alveolar macrophages.

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.

Defective Protein Prenylation in a Spectrum of Patients With Mevalonate Kinase Deficiency.

The rare autoinflammatory disease mevalonate kinase deficiency (MKD, which includes HIDS and mevalonic aciduria) is caused by recessive, pathogenic variants in the MVK gene encoding mevalonate kinase. Deficiency of this enzyme decreases the synthesis of isoprenoid lipids and thus prevents the normal post-translational prenylation of small GTPase proteins, which then accumulate in their unprenylated form. We recently optimized a sensitive assay capable of detecting unprenylated Rab GTPase proteins in peripheral blood mononuclear cells (PBMCs) and showed that this assay distinguished MKD from other autoinflammatory diseases. We have now analyzed PBMCs from an additional six patients with genetically-confirmed MKD (with different compound heterozygous MVK genotypes), and compared these with PBMCs from three healthy volunteers and four unaffected control individuals heterozygous for the commonest pathogenic variant, MVKV377I . We detected a clear accumulation of unprenylated Rab proteins, as well as unprenylated Rap1A by western blotting, in all six genetically-confirmed MKD patients compared to heterozygous controls and healthy volunteers. Furthermore, in the three subjects for whom measurements of residual mevalonate kinase activity was available, enzymatic activity inversely correlated with the extent of the defect in protein prenylation. Finally, a heterozygous MVKV377I patient presenting with autoinflammatory symptoms did not have defective prenylation, indicating a different cause of disease. These findings support the notion that the extent of loss of enzyme function caused by biallelic MVK variants determines the severity of defective protein prenylation, and the accumulation of unprenylated proteins in PBMCs may be a sensitive and consistent biomarker that could be used to aid, or help rule out, diagnosis of MKD.

Quantitative assays for measuring human telomerase activity and DNA binding properties.

Telomerase is the ribonucleoprotein enzyme that catalyzes the processive addition of the telomeric DNA repeat 5'-TTAGGG-3' onto chromosome ends. In addition to its fascinating biochemical and enzymatic properties, clinical interest in telomerase stems from its dysregulated expression in ∼90% of human cancers, representing a broad spectrum of diseases. Exploiting telomerase as a therapeutic target and hence identifying and/or evaluating potential inhibitors requires quantitative measurement of its activity. This article presents procedures for measuring multiple aspects of telomerase enzymology that are relevant to both fundamental biochemistry and drug discovery: direct activity assays, DNA binding affinity, DNA dissociation, and cell-based over-expression of the active enzyme complex.

Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.

Mevalonate kinase deficiency (MKD) is caused by mutations in a key enzyme of the mevalonate-cholesterol biosynthesis pathway, leading to recurrent autoinflammatory disease characterised by enhanced release of interleukin-1ß (IL-1ß). It is currently believed that the inflammatory phenotype of MKD is triggered by temperature-sensitive loss of mevalonate kinase activity and reduced biosynthesis of isoprenoid lipids required for the prenylation of small GTPase proteins. However, previous studies have not clearly shown any change in protein prenylation in patient cells under normal conditions. With lymphoblast cell lines from two compound heterozygous MKD patients, we used a highly sensitive in vitro prenylation assay, together with quantitative mass spectrometry, to reveal a subtle accumulation of unprenylated Rab GTPases in cells cultured for 3 days or more at 40 °C compared with 37 °C. This included a 200% increase in unprenylated Rab7A, Rab14 and Rab1A. Inhibition of sterol regulatory element-binding protein (SREBP) activation by fatostatin led to more pronounced accumulation of unprenylated Rab proteins in MKD cells but not parent cells, suggesting that cultured MKD cells may partially overcome the loss of isoprenoid lipids by SREBP-mediated upregulation of enzymes required for isoprenoid biosynthesis. Furthermore, while inhibition of Rho/Rac/Rap prenylation promoted the release of IL-1ß, specific inhibition of Rab prenylation by NE10790 had no effect in human peripheral blood mononuclear cells or human THP-1 monocytic cells. These studies demonstrate for the first time that mutations in mevalonate kinase can lead to a mild, temperature-induced defect in the prenylation of small GTPases, but that loss of prenylated Rab GTPases is not the cause of enhanced IL-1ß release in MKD.

A highly sensitive prenylation assay reveals in vivo effects of bisphosphonate drug on the Rab prenylome of macrophages outside the skeleton.

Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotropic effects outside the skeleton, most likely via cells of the monocyte/macrophage lineage exposed to nanomolar circulating drug concentrations. However, no effects of such low concentrations of ZOL have been reported using existing approaches. We have optimized a highly sensitive in vitro prenylation assay utilizing recombinant geranylgeranyltransferases to enable the detection of subtle effects of ZOL on the prenylation of Rab- and Rho-family GTPases. Using this assay, we found for the first time that concentrations of ZOL as low as 10nM caused inhibition of Rab prenylation in J774 macrophages following prolonged cell culture. By combining the assay with quantitative mass spectrometry we identified an accumulation of 18 different unprenylated Rab proteins in J774 cells after nanomolar ZOL treatment, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Finally, we also detected a clear effect of subcutaneous ZOL administration in vivo on the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages, confirming that systemic treatment with bisphosphonate drug can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo.

Real-time intravital imaging establishes tumor-associated macrophages as the extraskeletal target of bisphosphonate action in cancer.

UNLABELLED: Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs. SIGNIFICANCE: Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.

Direct involvement of the TEN domain at the active site of human telomerase.

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer K(d), supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5'-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3'-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.

Polarised secretion of cytokines in primary human microvascular endothelial cells is not dependent on N-linked glycosylation.

Human microvascular endothelial cells (HMVEC) grow in monolayers on Transwell filters and restrict permeability between the apical and basolateral media. We show that these cell monolayers are capable of sorting labelled endogenous proteins, including chemokines, growth factors and cytokines, to either the apical or basolateral media. IL-8 and GMCSF were secreted predominantly into the apical medium, whereas MIC-1 was secreted into the basolateral medium. This polarity did not correlate with glycosylation, as IL-8 and MIC-1 are both N-glycosylated, but were sorted to opposite sides of the cell. IL-6 is not glycosylated and did not display significant polarity in secretion. Similarly, the polarity of secretion of endogenous glycoproteins was not related to their glycosylation.