Two γ-zeins induce the unfolded protein response.

The rapid, massive synthesis of storage proteins that occurs during seed development stresses endoplasmic reticulum (ER) homeostasis, which activates the ER unfolded protein response (UPR). However, how different storage proteins contribute to UPR is not clear. We analyzed vegetative tissues of transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the common bean (Phaseolus vulgaris) soluble vacuolar storage protein PHASEOLIN (PHSL) or maize (Zea mays) prolamins (27-kDa γ-zein or 16-kDa γ-zein) that participate in forming insoluble protein bodies in the ER. We show that 16-kDa γ-zein significantly activates the INOSITOL REQUIRING ENZYME1/BASIC LEUCINE ZIPPER 60 (bZIP60) UPR branch-but not the bZIP28 branch or autophagy-leading to induction of major UPR-controlled genes that encode folding helpers that function inside the ER. Protein blot analysis of IMMUNOGLOBULIN-BINDING PROTEIN (BIP) 1 and 2, BIP3, GLUCOSE REGULATED PROTEIN 94 (GRP94), and ER-localized DNAJ family 3A (ERDJ3A) polypeptides confirmed their higher accumulation in the plant expressing 16-kDa γ-zein. Expression of 27-kDa γ-zein significantly induced only BIP3 and ERDJ3A transcription even though an increase in GRP94 and BIP1/2 polypeptides also occurred in this plant. These results indicate a significant but weaker effect of 27-kDa γ-zein compared to 16-kDa γ-zein, which corresponds with the higher availability of 16-kDa γ-zein for BIP binding, and indicates subtle protein-specific modulations of plant UPR. None of the analyzed genes was significantly induced by PHSL or by a mutated, soluble form of 27-kDa γ-zein that traffics along the secretory pathway. Such variability in UPR induction may have influenced the evolution of storage proteins with different tissue and subcellular localization.

Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana.

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

The WD40-repeat proteins NFC101 and NFC102 regulate different aspects of maize development through chromatin modification.

The maize (Zea mays) nucleosome remodeling factor complex component101 (nfc101) and nfc102 are putative paralogs encoding WD-repeat proteins with homology to plant and mammalian components of various chromatin modifying complexes. In this study, we generated transgenic lines with simultaneous nfc101 and nfc102 downregulation and analyzed phenotypic alterations, along with effects on RNA levels, the binding of NFC101/NFC102, and Rpd3-type histone deacetylases (HDACs), and histone modifications at selected targets. Direct NFC101/NFC102 binding and negative correlation with mRNA levels were observed for indeterminate1 (id1) and the florigen Zea mays CENTRORADIALIS8 (ZCN8), key activators of the floral transition. In addition, the abolition of NFC101/NFC102 association with repetitive sequences of different transposable elements (TEs) resulted in tissue-specific upregulation of nonpolyadenylated RNAs produced by these regions. All direct nfc101/nfc102 targets showed histone modification patterns linked to active chromatin in nfc101/nfc102 downregulation lines. However, different mechanisms may be involved because NFC101/NFC102 proteins mediate HDAC recruitment at id1 and TE repeats but not at ZCN8. These results, along with the pleiotropic effects observed in nfc101/nfc102 downregulation lines, suggest that NFC101 and NFC102 are components of distinct chromatin modifying complexes, which operate in different pathways and influence diverse aspects of maize development.

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin.

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Expression of CLAVATA3 fusions indicates rapid intracellular processing and a role of ERAD.

The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum chaperone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

Altered skeletal muscle subsarcolemmal mitochondrial compartment during catch-up fat after caloric restriction.

An accelerated rate of fat recovery (catch-up fat) and insulin resistance are characteristic features of weight recovery after caloric restriction, with implications for the pathophysiology of catch-up growth and weight fluctuations. Using a previously described rat model of weight recovery in which catch-up fat and skeletal muscle insulin resistance have been linked to suppressed thermogenesis per se, we investigated alterations in mitochondrial energetics and oxidative stress in subsarcolemmal (SS) and intermyofibrillar (IMF) skeletal muscle mitochondria. After 2 weeks of semistarvation followed by 1 week of refeeding, the refed rats show persistent and selective reductions in SS mitochondrial mass (assessed from citrate synthase activity in tissue homogenate and isolated mitochondria) and oxidative capacity. Furthermore, the refed rats show, in both SS and IMF muscle mitochondria, a lower aconitase activity (whose inactivation is an index of increased reactive oxygen species [ROS]), associated with higher superoxide dismutase activity and increased proton leak. Taken together, these studies suggest that diminished skeletal muscle mitochondrial mass and function, specifically in the SS mitochondrial compartment, contribute to the high metabolic efficiency for catch-up fat after caloric restriction and underscore a potential link between diminished skeletal muscle SS mitochondrial energetics, increased ROS concentration, and insulin resistance during catch-up fat.

A role for skeletal muscle stearoyl-CoA desaturase 1 in control of thermogenesis.

An enhanced metabolic efficiency for accelerating the recovery of fat mass (or catch-up fat) is a characteristic feature of body weight regulation after weight loss or growth retardation and is the outcome of an "adipose-specific" suppression of thermogenesis, i.e., a feedback control system in which signals from the depleted adipose tissue fat stores exert a suppressive effect on thermogenesis. Using a previously described rat model of semistarvation-refeeding in which catch-up fat results from suppressed thermogenesis per se, we report here that the gene expression of stearoyl-coenzyme A desaturase 1 (SCD1) is elevated in skeletal muscle after 2 wk of semistarvation and remains elevated in parallel to the phase of suppressed thermogenesis favoring catch-up fat during refeeding. These elevations in the SCD1 transcript are skeletal muscle specific and are associated with elevations in microsomal Delta9 desaturase enzyme activity, in the Delta9 desaturation index, and in the relative content of SCD1-derived monounsaturates in several lipid fractions extracted from skeletal muscle. An elevated skeletal muscle SCD1, by desaturating the products of de novo lipogenesis and diverting them away from mitochondrial oxidation, would inhibit substrate cycling between de novo lipogenesis and lipid oxidation, thereby leading to a state of suppressed thermogenesis that regulates the body's fat stores.

Redistribution of glucose from skeletal muscle to adipose tissue during catch-up fat: a link between catch-up growth and later metabolic syndrome.

Catch-up growth, a risk factor for later obesity, type 2 diabetes, and cardiovascular diseases, is characterized by hyperinsulinemia and an accelerated rate for recovering fat mass, i.e., catch-up fat. To identify potential mechanisms in the link between hyperinsulinemia and catch-up fat during catch-up growth, we studied the in vivo action of insulin on glucose utilization in skeletal muscle and adipose tissue in a previously described rat model of weight recovery exhibiting catch-up fat caused by suppressed thermogenesis per se. To do this, we used euglycemic-hyperinsulinemic clamps associated with the labeled 2-deoxy-glucose technique. After 1 week of isocaloric refeeding, when body fat, circulating free fatty acids, or intramyocellular lipids in refed animals had not yet exceeded those of controls, insulin-stimulated glucose utilization in refed animals was lower in skeletal muscles (by 20-43%) but higher in white adipose tissues (by two- to threefold). Furthermore, fatty acid synthase activity was higher in adipose tissues from refed animals than from fed controls. These results suggest that suppressed thermogenesis for the purpose of sparing glucose for catch-up fat, via the coordinated induction of skeletal muscle insulin resistance and adipose tissue insulin hyperresponsiveness, might be a central event in the link between catch-up growth, hyperinsulinemia and risks for later metabolic syndrome.

Where do Protein Bodies of Cereal Seeds Come From?

Protein bodies of cereal seeds consist of ordered, largely insoluble heteropolymers formed by prolamin storage proteins within the endoplasmic reticulum (ER) of developing endosperm cells. Often these structures are permanently unable to traffic along the secretory pathway, thus representing a unique example for the use of the ER as a protein storage compartment. In recent years, marked progress has been made in understanding what is needed to make a protein body and in formulating hypotheses on how protein body formation might have evolved as an efficient mechanism to store large amounts of protein during seed development, as opposed to the much more common system of seed storage protein accumulation in vacuoles. The major key evolutionary events that have generated prolamins appear to have been insertions or deletions that have disrupted the conformation of the eight-cysteine motif, a protein folding motif common to many proteins with different functions and locations along the secretory pathway, and, alternatively, the fusion between the eight-cysteine motif and domains containing additional cysteine residues.

A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco.

Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

The direct effect of leptin on skeletal muscle thermogenesis is mediated by substrate cycling between de novo lipogenesis and lipid oxidation.

We report here studies that integrate data of respiration rate from mouse skeletal muscle in response to leptin and pharmacological interference with intermediary metabolism, together with assays for phosphatidylinositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK). Our results suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle is mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that this cycle requires both PI3K and AMPK signaling. This substrate cycling linking glucose and lipid metabolism to thermogenesis provides a novel thermogenic mechanism by which leptin protects skeletal muscle from excessive fat storage and lipotoxicity.

Protein body formation in the endoplasmic reticulum as an evolution of storage protein sorting to vacuoles: insights from maize γ-zein.

The albumin and globulin seed storage proteins present in all plants accumulate in storage vacuoles. Prolamins, which are the major proteins in cereal seeds and are present only there, instead accumulate within the endoplasmic reticulum (ER) lumen as very large insoluble polymers termed protein bodies. Inter-chain disulfide bonds play a major role in polymerization and insolubility of many prolamins. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body formation when fused to other proteins and contains seven cysteine residues involved in inter-chain bonds. We show that progressive substitution of these amino acids with serine residues in full length γ-zein leads to similarly progressive increase in solubility and availability to traffic from the ER along the secretory pathway. Total substitution results in very efficient secretion, whereas the presence of a single cysteine is sufficient to promote partial sorting to the vacuole via a wortmannin-sensitive pathway, similar to the traffic pathway of vacuolar storage proteins. We propose that the mechanism leading to accumulation of prolamins in the ER is a further evolutionary step of the one responsible for accumulation in storage vacuoles.

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

Skeletal muscle mitochondrial efficiency and uncoupling protein 3 in overeating rats with increased thermogenesis.

To establish whether changes in skeletal muscle mitochondrial efficiency contribute to increased energy expenditure and decreased metabolic efficiency of overeating rats with increased thermogenesis, we measured basal proton leak, fatty acid-induced uncoupling and uncoupling protein 3 (UCP3) content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Intermyofibrillar, but not subsarcolemmal, mitochondria from rats with increased thermogenesis exhibited a lower proton leak compared with controls. In both mitochondrial populations from rats with increased thermogenesis, fatty acid-induced uncoupling was increased significantly and a small recoupling effect of GDP was detected. In addition, intermyofibrillar and subsarcolemmal mitochondria from rats with increased thermogenesis showed higher UCP3 contents than controls. These results point out that metabolic efficiency in subsarcolemmal and intermyofibrillar mitochondria from rats with increased thermogenesis is differently regulated. In fact, in intermyofibrillar mitochondria both basal proton leak and fatty acid-induced uncoupling are altered, while in subsarcolemmal mitochondria only fatty acid-induced uncoupling increases. Both mitochondrial populations in skeletal muscle cells from rats with increased thermogenesis display an increased fatty acid-induced uncoupling and UCP3 content, which could contribute to avoiding obesity.

Zeolin. A new recombinant storage protein constructed using maize gamma-zein and bean phaseolin.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of gamma-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type gamma-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the gamma-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.