The Small, Methionine-Rich Chloroplast Heat-Shock Protein Protects Photosystem II Electron Transport during Heat Stress

Evidence suggests that the small chloroplast heat-shock protein (Hsp) is involved in plant thermotolerance but its site of action is unknown. Functional disruption of this Hsp using anti-Hsp antibodies or addition of purified Hsp to chloroplasts indicated that (a) this Hsp protects thermolabile photosystem II and, consequently, whole-chain electron transport during heat stress; and (b) this Hsp completely accounted for heat acclimation of electron transport in pre-heat-stressed plants. Therefore, this Hsp is a major adaptation to acute heat stress in plants.     

Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1^-/- male mice have normal fertility, Tpst2^-/- males are infertile despite normal spermatogenesis. However, Tpst2^-/- sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1^-/-, but not in Tpst2^-/- mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- males.Protein-tyrosine sulfation is a post-translational modification described over 50 years ago (1). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase activity have been described in many species in the plant and animal kingdoms (2, 3). In humans, dozens of tyrosine-sulfated proteins have been identified. These include certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, hormones, and others. It has been demonstrated that some of these proteins require tyrosine sulfation for optimal function (3).In mice and humans, protein-tyrosine sulfation is mediated by one of two tyrosylprotein sulfotransferases called TPST-1² and TPST-2 (4–6). Mouse TPST-1 and TPST-2 are 370- and 376-residue type II transmembrane proteins, respectively. Each has a short N-terminal cytoplasmic domain followed by a single ≈17-residue transmembrane domain, a membrane proximal ≈40-residue stem region, and a luminal catalytic domain containing four conserved Cys residues and two N-glycosylation sites. The amino acid sequence of human and mouse TPST-1 are ≈96% identical and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ≈65–67% identical to TPST-2 in both mice and humans. TPST-1 and TPST-2 are broadly expressed in human and murine tissues and cell lines and are co-expressed in most, if not all, cell types (3).A variety of biochemical studies have shown that protein-tyrosine sulfation occurs exclusively in the trans-Golgi network (7, 8). This conclusion has been strengthened by more recent immunofluorescence studies showing that a TPST-1/enhanced green fluorescent protein fusion protein co-localizes with golgin-97, a marker for the trans-Golgi network (9). Thus, protein-tyrosine sulfation occurs only on proteins that transit the secretory pathway and occurs well after protein folding and disulfide formation are complete and after N- and O-linked glycosylation are initiated.To gain an understanding of the biological importance of TPSTs, we have generated TPST-deficient mice by targeted disruption of either the Tpst1 or Tpst2 gene. Our studies of Tpst1^-/- mice revealed unexpected but modest effects on body weight and fecundity (10). Tpst1^-/- mice appear healthy but have ≈5% lower average body weight than wild type mice. Fertility of Tpst1^-/- males and females per se was normal. However, Tpst1^-/- females have smaller litters than wild type females due to embryonic lethality between 8.5 and 15.5 days post coitum.In our studies of Tpst2^-/- mice we found that Tpst2^-/- males were infertile, in contrast to Tpst1^-/- males that have normal fertility (11). We found that Tpst2^-/- males were eugonadal and have normal spermatogenesis. Epididymal sperm from Tpst2^-/- males were normal in number, morphology, and motility and appeared to capacitate in vitro and undergo acrosome exocytosis in response to agonist. However, Tpst2^-/- sperm are severely defective in motility in viscous media and in their ability to fertilize zona pellucida (ZP)-intact eggs. In addition, in vitro fertilization experiments revealed that Tpst2^-/- sperm had reduced ability to adhere to the egg plasma membrane, but were able to undergo membrane fusion with the egg.These findings suggest that tyrosine sulfation of one or more substrates is crucial for normal sperm function. However, there are no proteins directly involved in sperm function that are known to be tyrosine-sulfated. The luteinizing hormone receptor and follicle-stimulating hormone receptor are the only proteins important in reproductive biology that are known to be tyrosine-sulfated. Both receptors have been shown to be sulfated at a membrane proximal site in their respective N-terminal extracellular domains that are conserved in many species including the mouse (12). Sulfation of these receptors has been shown to be required for optimal affinity of their cognate ligands in vitro. However, our observations that serum LH, FSH, and testosterone levels are normal in Tpst2^-/- males coupled with the observation that spermatogenesis is normal excludes defective sulfation of these receptors as an explanation for infertility of Tpst2^-/- males (11).In this study, we sought to identify tyrosine-sulfated proteins expressed in the male genital tract that may provide clues as to the mechanism for the infertility of Tpst2^-/- male mice. Among the several candidate tyrosine-sulfated proteins that were identified, RNase 9 and Mfge8 were of particular interest. RNase 9 is a catalytically inactive RNase A family member of unknown function and is expressed only in the epididymis after onset of sexual maturity (13). Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice have been reported to be subfertile (14). Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated. We also showed that both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides, and that RNase 9 and Mfge8 are sulfated in wild type and Tpst1^-/- mice, but not in Tpst2^-/- mice. Therefore, lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- male mice.     

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [¹⁴C]2,4-dichlorophenoxyacetic acid and [³H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [³H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [¹⁴C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Small Hydrophobic Protein of Human Metapneumovirus Does Not Affect Virus Replication and Host Gene Expression In Vitro

Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.     

Membrane-type 1 Matrix Metalloproteinase Regulates Macrophage-dependent Elastolytic Activity and Aneurysm Formation in Vivo

During arterial aneurysm formation, levels of the membrane-anchored matrix metalloproteinase, MT1-MMP, are elevated dramatically. Although MT1-MMP is expressed predominately by infiltrating macrophages, the roles played by the proteinase in abdominal aortic aneurysm (AAA) formation in vivo remain undefined. Using a newly developed chimeric mouse model of AAA, we now demonstrate that macrophage-derived MT1-MMP plays a dominant role in disease progression. In wild-type mice transplanted with MT1-MMP-null marrow, aneurysm formation induced by the application of CaCl₂ to the aortic surface was almost completely ablated. Macrophage infiltration into the aortic media was unaffected by MT1-MMP deletion, and AAA formation could be reconstituted when MT1-MMP^+/+ macrophages, but not MT1-MMP^+/+ lymphocytes, were infused into MT1-MMP-null marrow recipients. In vitro studies using macrophages isolated from either WT/MT1-MMP^-/- chimeric mice, MMP-2-null mice, or MMP-9-null mice demonstrate that MT1-MMP alone plays a dominant role in macrophage-mediated elastolysis. These studies demonstrate that destruction of the elastin fiber network during AAA formation is dependent on macrophage-derived MT1-MMP, which unexpectedly serves as a direct-acting regulator of macrophage proteolytic activity.Development and progression of abdominal aortic aneurysm (AAA)² is a complex process that, untreated, can lead to tissue failure, hemorrhage, and death (1). Destruction of the orderly elastin lamellae of the vessel wall is considered the sine qui non of arterial aneurysm formation (2) as adult tissues cannot regenerate normal elastin fibers (3). Moreover, the elastin degradation products are chemotactic for inflammatory cells and serve to amplify the local injury (4). Although several types of elastolytic proteases are elevated in AAA tissue (5-9), studies using murine models of AAA and targeted protease deletion suggest that matrix metalloproteinases (MMPs), particularly the secreted proteases, MMP-2 and MMP-9, play key roles in the pathologic remodeling of the elastin lamellae that lead to AAA (7, 8).Membrane-type 1 MMP (MT1-MMP) is the prototypical member of a family of membrane-tethered MMPs (10). Recent studies indicate that MT1-MMP expression is elevated in human AAA tissues and that infiltrating macrophages are the primary source of the proteinase in aortic lesions (11-13). Although indirect evidence suggests that MT1-MMP may participate in the control of monocyte/macrophage motile responses as well as interactions with the vessel wall during transmigration (14, 15), the role(s) played by MT1-MMP in regulating macrophage proteolytic activity or AAA formation in vivo remains undefined.Using a murine model of AAA and mice with a targeted deletion of MT1-MMP in myelogenous cell populations, we now demonstrate that macrophage-derived MT1-MMP is required for elastin degradation and aneurysm formation. Importantly, macrophages are not dependent on MT1-MMP for infiltrating aortic tissues but instead use the protease to directly regulate their elastolytic potential in an MMP-2- and MMP-9-independent fashion. These studies define a new and unexpected role for MT1-MMP in controlling macrophage elastolytic activity in the in vitro and in vivo settings.     

In Vivo Substrate Diversity and Preference of Small Heat Shock Protein IbpB as Revealed by Using a Genetically Incorporated Photo-cross-linker

Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones found in all forms of life, are known to be able to protect cells against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro conditions. Nevertheless, it is poorly understood what natural substrate proteins are protected by sHSPs in living cells. Here, by using a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we identified a total of 95 and 54 natural substrate proteins of IbpB (an sHSP from Escherichia coli) in living cells with and without heat shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins (e.g. ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic enzymes. Furthermore, these two classes of proteins were found to be more prone to aggregation and/or inactivation in cells lacking IbpB under stress conditions (e.g. heat shock). Together, our in vivo data offer novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential protection on the protein synthesis machine and metabolic enzymes may dominantly contribute to the well known protective effect of sHSPs on cell survival against stresses.     

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell’s environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.     

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl₂ = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.     

The Induction of Lipocalin-2 Protein Expression in Vivo and in Vitro

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.     

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10–17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.Organisms use color vision for survival behaviors such as foraging, mating, and predator avoidance (1–3). Color vision in invertebrates ranges from trichromatic systems capable of detecting UV, blue, and green (e.g. bees and flies) to the highly complex mantis shrimps (stomatopods) having 12 spectrally distinct classes of photoreceptor cells (4). Despite the diversity of invertebrate color vision systems and the large collection of naturally occurring visual pigments, important questions remain concerning the molecular mechanisms that regulate color sensitivity.In both vertebrates and invertebrates, the visual pigment rhodopsin consists of a chromophore (e.g. 11-cis retinal) covalently bound to an opsin apoprotein via a protonated Schiff base. Upon light absorption, the chromophore isomerizes from cis to all-trans, inducing conformational changes in the opsin that produce activated metarhodopsin. Specific interactions between the retinal chromophore and residues in the opsin tune the λ_max of the chromophore. Studies have shown that Glu-113 (bovine position) serves as the retinylidene Schiff base counter-ion in vertebrate visual pigments (5–7). Removing the negative charge of the counter-ion from the binding pocket deprotonates the chromophore and yields a UV-absorbing pigment (5–7). Using sequence alignments, phylogenetic analysis, analysis of the bovine rhodopsin crystal structure (PDB² entry 1U19), and functional experiments, a large number of amino acids involved in the spectral tuning of vertebrate visual pigments have been identified (8).In contrast, the counter-ion for invertebrate rhodopsin remains unknown, and only one spectrally relevant residue has been identified: an amino acid substitution in Drosophila pigments responsible for UV versus visible sensitivity (9). Interestingly, this amino acid substitution (Gly-90 in bovine rhodopsin) coincides with a substitution that mediates UV versus blue sensitivity in several bird species (10, 11). This discovery highlights the value of a cross-phyla comparison of visual pigments as a means to identify structural differences that may regulate color vision in invertebrates.In the present study, we identify an amino acid substitution in Drosophila visual pigments that regulates the color sensitivity of blue- and green-absorbing rhodopsins. For these studies, we employed sequence analysis of invertebrate and vertebrate visual pigments and a functional examination of mutant invertebrate opsins. This amino acid substitution red-shifts the λ_max of the Drosophila Rh1 pigment and reciprocally blue-shifts the λ_max of Rh6 pigment. Interestingly, this site also affects the spectral tuning of vertebrate pigments and corresponds to Ala-292 in bovine rhodopsin (8, 12–16).