Liam J. Murphy

The Prohibitins: emerging roles in diverse functions

The prohibitins, Phb1 and Phb2 are highly conserved proteins in eukaryotic cells that are present in multiple cellular compartments. Initial investigations focused on the role of Phb1 as an inhibitor of cell proliferation hence the original name prohibitin. However both proteins appear to have a diverse range of functions and recent evidence suggests that the prohibitins have very similar but as yet only partially understood functions. In addition to their role as chaperone proteins in the mitochondria, and their ability to target to lipid rafts, their is now compelling evidence that both prohibitins are localized in the nucleus and can modulate transcriptional activity by interacting with various transcription factors, including the steroid hormone receptors, either directly or indirectly. In addition Phb1 and Phb2 are present in the circulation and can be internalized when added to cultured cells suggesting that the circulating prohibitins may have some regulatory role. This review presents some of the recent developments in prohibitin research and focuses on the similarities in the structure and function of these interesting proteins.

A Schwann cell mitogen accompanying regeneration of motor neurons

Motor neurons are the only adult mammalian neurons of the central nervous system to regenerate following injury. This ability is dependent on the environment of the peripheral nerve and an intrinsic capacity of motor neurons for regrowth. We report here the identification, using a technique known as messenger RNA differential display, of an extracellular signalling molecule, previously described as the pancreatic secreted protein Reg-2 (ref. 4), that is expressed solely in regenerating and developing rat motor and sensory neurons. Axon-stimulated Schwann cell proliferation is necessary for successful regeneration,, and we show that Reg-2 is a potent Schwann cell mitogen in vitro. In vivo, Reg-2 protein is transported along regrowing axons and inhibition of Reg-2 signalling significantly retards the regeneration of Reg-2-containing axons. During development, Reg-2 production by motor and sensory neurons is regulated by contact with peripheral targets. Strong candidates for peripheral factors regulating Reg-2 production are cytokines of the LIF/CNTF family, because Reg-2 is not expressed in developing motor or sensory neurons of mice carrying a targeted disruption of the LIF receptor gene, a common component of the receptor complexes for all of the LIF/CNTF family.

Estrogen receptor-β mRNA variants in human and murine tissues

Abstract Estrogen receptor (ER)- β mRNA splice variants have been identified in human breast tumors as well as normal human and mouse ovarian, uterine and mammary tissues. In both species transcripts deleted in exons 5 or 6, or 5+6 have been characterized by RT-PCR followed by cloning and sequencing. In mouse tissues an ER- β transcript containing 54 nucleotides inserted in frame between exons 5 and 6 was identified. Interestingly, no equivalent of the mouse inserted transcript was detected in any of the four human tissues analyzed.

Increased Renal Aldose Reductase Activity, Immunoreactivity, and mRNA in Streptozocin-Induced Diabetic Rats

Increased accumulation of renal sorbitol has been documented in the diabetic rat, and it has been suggested that this accumulation may be important in the pathogenesis of diabetic nephropathy. It is not clear whether sorbitol accumulation results from increases in substrate, activity of the aldose reductase (AR) protein molecule, or activity due to an increase in the amount of enzyme present. In this study, we have quantitated renal AR activity, immunoreactivity, and mRNA in rats 3 mo after induction of diabetes with streptozocin (STZ-D, 65 mg/kg body wt). Renal AR activity was significantly increased in diabetic rats compared with age-matched nondiabetic controls (0.95 ± 0.05 vs. 0.51 ± 0.03 U · mg−1 · h−1, respectively, P < .0005). Western blot analysis demonstrated that the antiserums recognized a single 40,000-Mr, protein species in renal homogenates from both diabetic and nondiabetic rats. When quantitated in an immunodot assay, AR immunoreactivity was significantly increased in diabetic rats compared with nondiabetic controls (0.57 ± 0.03 vs. 0.33 ± 0.02 U, respectively, P < .0005). Hybridization of Northern blots with a synthetic 36-nucleotide oligomer and an AR cDNA identified a 1.4-kilobase pair transcript; the abundance of the transcript was significantly increased in poly(A)+RNA from the kidneys of diabetic compared with nondiabetic rats (P < .005). This study demonstrates that renal AR activity is increased in the STZ-D rats and suggests that the increased AR activity can be in part explained by enhanced AR gene expression.

Perturbations in bone formation and resorption in insulin-like growth factor binding protein-3 transgenic mice.

IGF‐I and their binding proteins are important in bone health. Examination of BMD, osteoblast proliferation, and markers of bone resorption in transgenic mice that constitutively overexpress IGFBP‐3 indicates that overexpression of IGFBP‐3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation.

Acarbose in the treatment of elderly patients with type 2 diabetes.

AIMS To study the effect of acarbose, an alpha-glucosidase inhibitor, on glycemic control in elderly patients with type 2 diabetes. METHODS Elderly patients with type 2 diabetes treated with diet alone were randomly treated in a double-blind fashion with placebo (n=99) or acarbose (n=93) for 12 months. RESULTS After 12 months of therapy, there was a statistically significant difference in the change in glycated haemoglobin (HbA(1c)) (-0.6%) in the acarbose group versus placebo, as well as in the incremental post-prandial glucose values (-2.1 mmol h/l) and mean fasting plasma glucose (-0.7 mmol/l). Although there was no effect of acarbose on insulin release, there was a clear effect of acarbose to decrease relative insulin resistance (-0.8) (HOMA method). In addition, acarbose was generally well tolerated and safe in the elderly; most discontinuations were due to gastrointestinal side effects such as flatulence and diarrhea. There were no cases of hypoglycemia reported, and no clinically relevant changes in laboratory abnormalities or vital signs during the study. CONCLUSIONS Acarbose improves the glycemic profile and insulin sensitivity in elderly patients with type 2 diabetes who are inadequately controlled on diet alone.

Phosphorylation of histones by tissue transglutaminase

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser10 in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser10 by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17β-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.

The effects of GH replacement in adult GH‐deficient patients: changes in body composition without concomitant changes in the adipokines and insulin resistance

background  Growth hormone deficiency (GHD) in adult life has been associated with increased central adiposity, decreased insulin sensitivity, dyslipidaemia and increased risk of cardiovascular disease. The effects of GH replacement on adiponectin and resistin, adipokines which have a role in modulating insulin sensitivity have not been previously reported.

Perturbations in adiponectin, leptin and resistin levels in acromegaly: lack of correlation with insulin resistance

background Insulin resistance, impaired glucose tolerance and type 2 diabetes are common in acromegalic subjects. The mechanism underlying this insulin resistance is unclear.

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein.

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb·E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2–/– cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.