From endoplasmic reticulum to secretory granules: role of zinc in the secretory pathway of growth hormone

Endocrine and neuroendocrine cells differ from cells which rapidly release all their secreted proteins in that they store some secretory proteins in concentrated forms in secretory granules to be rapidly released when cells are stimulated. Protein aggregation is considered as the first step in the secretory granule biosynthesis and, at least in the case of prolactin and growth hormone, greatly depends on zinc ions that facilitate this process. Hence, regulation of cellular zinc transport especially that within the regulated secretory pathway is of importance to understand. Various zinc transporters of Slc30a/ZnT and Slc39a/Zip families have been reported to fulfil this role and to participate in fine tuning of zinc transport in and out of the endoplasmic reticulum, Golgi complex and secretory granules, the main cellular compartments of the regulated secretory pathway. In this review, we will focus on the role of zinc in the formation of hormone-containing secretory granules with special emphasis on conditions required for growth hormone dimerization/aggregation. In addition, we highlight the role of zinc transporters that govern the process of zinc homeostasis in the regulated hormone secretion.

Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli

Translational pausing may occur due to a number of mechanisms, including the presence of non-optimal codons, and it is thought to play a role in the folding of specific polypeptide domains during translation and in the facilitation of signal peptide recognition during see-dependent protein targeting. In this whole genome analysis of Escherichia coli we have found that non-optimal codons in the signal peptide-encoding sequences of secretory genes are overrepresented relative to the mature portions of these genes; this is in addition to their overrepresentation in the 5'-regions of genes encoding non-secretory proteins. We also find increased non-optimal codon usage at the 3' ends of most E. coli genes, in both non-secretory and secretory sequences. Whereas presumptive translational pausing at the 5' and 3' ends of E. coli messenger RNAs may clearly have a general role in translation, we suggest that it also has a specific role in sec-dependent protein export, possibly in facilitating signal peptide recognition. This finding may have important implications for our understanding of how the majority of non-cytoplasmic proteins are targeted, a process that is essential to all biological cells. (C) 2004 Elsevier Inc. All rights reserved.

Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.

Neurotrophins and neuronal plasticity in the action of antidepressants and morphine

Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.

Recovery of Yeast Saccharomyces cerevisiae after Thermal Insult

All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.

Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

Estudo da expressão e participação de osteopontina durante a interação taquizoíto de Toxoplasma gondii célula hospedeira.

Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras. .

Mechanisms of substrate selection by the signal recognition particle

The signal recognition particle (SRP) targets membrane and secretory proteins to their correct cellular destination with remarkably high fidelity. Previous studies have shown that multiple checkpoints exist within this targeting pathway that allows ‘correct cargo’ to be quickly and efficiently targeted and for ‘incorrect cargo’ to be promptly rejected. In this work, we delved further into understanding the mechanisms of how substrates are selected or discarded by the SRP. First, we discovered the role of the SRP fingerloop and how it activates the SRP and SRP receptor (SR) GTPases to target and unload cargo in response to signal sequence binding. Second, we learned how an ‘avoidance signal’ found in the bacterial autotransporter, EspP, allows this protein to escape the SRP pathway by causing the SRP and SR to form a ‘distorted’ complex that is inefficient in delivering the cargo to the membrane. Lastly, we determined how Trigger Factor, a co-translational chaperone, helps SRP discriminate against ‘incorrect cargo’ at three distinct stages: SRP binding to RNC; targeting of RNC to the membrane via SRP-FtsY assembly; and stronger antagonism of SRP targeting of ribosomes bearing nascent polypeptides that exceed a critical length. Overall, results delineate the rich underlying mechanisms by which SRP recognizes its substrates, which in turn activates the targeting pathway and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved by this protein targeting machinery.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibe[ carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium. (C) 2009 Elsevier Inc. All rights reserved.

The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8 - a novel prokineticin from Bombina orientalis

Prokineticins are small (8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum ( ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor ( EGFR) and plasminogen activator receptor ( uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells. Laboratory Investigation (2009) 89, 1229-1242; doi: 10.1038/labinvest.2009.87; published online 21 September 2009