Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca 2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions.

No abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells.

Although steroid-induced negative effects on bone and collagen have been well described in corticosteroid-treated patients, few studies have extensively evaluated bone and collagen turnover in patients with endogenous Cushing's syndrome. In this work serum bone-Gla protein (BGP), C-terminal cross-linked telopeptide of type I collagen (ICTP) and N-terminal propeptide of type III procollagen (PIIINP) levels were determined in patients with active (n ¼ 12) and preclinical (n ¼ 6) Cushing's syndrome, adrenal incidentalomas (n ¼ 35) and in healthy controls (n ¼ 28). In patients with overt Cushing's syndrome, serum BGP (0.9 Ϯ 0.2 ng/ml), ICTP (2.7 Ϯ 0.2 ng/ml) and PIIINP (1.9 Ϯ 0.2 ng/ml) levels were significantly lower (P < 0.0001) than in controls (5.5 Ϯ 0.2, 3.9 Ϯ 0.2 and 3.2 Ϯ 0.2 ng/ml respectively). In preclinical Cushing's syndrome, serum BGP (2.5 Ϯ 0.8 ng/ml), ICTP (2.2 Ϯ 0.1 ng/ml) and PIIINP (2.2 Ϯ 0.2 ng/ml) levels were significantly lower than in normal subjects (P < 0.0001, P < 0.0001 and P < 0.02 respectively), being similar to those recorded in overt Cushing's syndrome. In patients with adrenal incidentaloma, serum BGP (4.2 Ϯ 0.5 ng/ml) and ICTP (2.9 Ϯ 0.2 ng/ml) levels were significantly lower than those found in controls (P < 0.05 and P < 0.001 respectively), while serum PIIINP levels (3.6 Ϯ 0.2 ng/ml) did not differ from those of normals. In particular, 9/35 patients with adrenal incidentaloma had markedly depressed BGP levels (<2.0 ng/ml; mean 0.8 Ϯ 0.1 ng/ml); all patients of this subgroup showed an exaggerated 17-hydroxyprogesterone increase after ACTH administration. In the same patients, serum ICTP (3.0 Ϯ 0.4 ng/ml) and PIIINP (3.6 Ϯ 0.2 ng/ml) levels did not differ from those found in the incidentaloma group. In conclusion, our study indicates that bone and collagen turnover are markedly affected in patients with overt and preclinical Cushing's syndrome. Although patients with adrenal incidentaloma do not show any signs or symptoms of overt hypercortisolism, the presence of reduced BGP and ICTP levels might be considered a further index of an 'abnormal' pattern of steroid secretion in some of them. As a consequence, the presence of early alterations in markers of bone turnover might be useful for selecting those patients who need more accurate follow-up of the adrenal mass.