Snapshot Peptidomics of the Regulated Secretory Pathway

Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PC-mediated processing have been described ((R/K)X_n(R/K) where n = 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine α-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.The generation of peptide hormones or neuropeptides involves the proteolytic processing of precursor proteins by specific proteases. In neurons and endocrine cells, most, if not all, of these bioactive peptides are generated within the RSP¹ in which the processing enzymes PC1/3 or PC2 cleave precursors at basic residues (1, 2). The PC-mediated cleavage most often occurs at consecutive basic residues, but not all basic residues serve as PC recognition sites (2). This is partly because the secondary structure of a precursor also affects the substrate recognition (3). Identification of processing sites is hence a prerequisite for locating unknown peptides hidden in a precursor sequence.Peptidomics has been advocated to comprehensively study peptides cleaved off from precursor proteins by endogenous proteases (4–6). These naturally occurring peptides are beyond the reach of current proteomics and should be analyzed in their native forms. Unlike proteomics, peptidomics has the potential to uncover processing sites of precursor proteins. Most peptidomics studies, which target tissue peptidomes from brain or endocrine organs (7–11), have provided limited information about secretory peptides that could help to identify processing sites; they are too often blurred by subsequent actions of exopeptidases (cutting off a single amino acid or dipeptide from either end of a peptide).In MS-based identification of bioactive peptides present in biological samples, their relative low abundance in a total pool of naturally occurring peptides should be considered. Once extracted from cultured cells or tissues, bona fide secretory peptides and nonsecretory peptides or peptide fragments caused by degradation of abundant cytosolic proteins cannot be discriminated, and therefore we need to analyze samples rich in secretory peptides to facilitate the identification of bioactive peptides. Several attempts have been made to isolate secretory proteins or peptides, such as subcellular fractionation for harvesting secretory granules (12, 13). With all these efforts, a limited number of secretory peptides have been identified, and many known bioactive peptides still escape analysis.We took advantage of the fact that peptides processed in the RSP are enriched in secretory granules of neurons and endocrine cells and released on exocytosis. Here we applied a brief exocytotic stimulus (2 min) to cultured human endocrine cells and identified peptides released into supernatant using LC-MSMS on an LTQ-Orbitrap mass spectrometer. Nearly 97% of the identified peptides arose from precursor proteins known to be recruited to the RSP, such as peptide hormone precursors and granin-like secretory proteins. Our approach was validated by the identification of previously known processing sites of peptide hormone precursors. In addition, a majority of the identified peptides retained cleavage sites that agree with consensus cleavage sites for PCs, which are informative enough to deduce the processing sites of RSP proteins. This peptidomics approach will expedite the identification of unknown bioactive peptides.     

酵母蛋白分泌途径的研究进展

酵母表达系统是目前应用广泛的真核表达系统之一。本文基于对酵母的相关研究近况,分析了酵母体内的分泌途径,推测了可能影响酵母表达系统表达量的原因,并提出了可能解决的方法,为今后的工作打下基础。     

Membrane Dynamics in the Early Secretory Pathway

All eukaryotes possess a secretory pathway, and the major molecular players involved in secretion are well conserved. However, the morphological manifestation of this pathway at the level of the participant organelles shows great divergences between yeasts, mammals and plants. The unique features of the early secretory pathway in plants—a polydisperse mobile Golgi apparatus and the lack of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus—suggests the participation of many plant-specific molecules in the maintenance and regulation of protein trafficking. The advent of live cell imaging fluorescently-tagged proteins and the increased usage of cryotechniques in electron microscopy has led to dramatic advances in our understanding of the early secretory pathway of plants. In contrast, contradictions have sometimes emerged and interpretations for the same observations have not necessarily reached a consensus. In this review we have attempted to provide the reader with a critical, yet balanced overview of this rapidly expanding research area. Wherever possible we have contrasted a particular event or parameter with the corresponding situation in yeast or mammalian cells. We have also taken the opportunity to suggest suitable experimentation in newly emerging sectors.     

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.     

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA)² (2–6), where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7, 8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction “hub” according to their binding affinity.Quantitative estimation of protein-protein interactions in vivo will require the amount of the complex to be directly measured or the level of reconstituted reporters to be directly proportional to the strength of the interactions. To achieve quantitative analysis of protein interactions in eukaryotic expression system, we have designed a yeast surface two-hybrid (YS2H) system that can express a pair of proteins, one protein as a fusion to a yeast cell wall protein, agglutinin, and the other in a secretory form. When two proteins interact in this system, they associate in the secretory pathway, and the prey that would otherwise be released into the media is captured on the surface by the bait. We have devised two different schemes to quantitatively estimate the affinity of two interacting molecules: flow cytometric detection of antibody binding to the epitope tags fused to the prey and the bait, and the GFP readout from the complementation of split GFP fragments fused to the prey and the bait. They are induced under a bi-directional promoter to promote a synchronized and comparable level of expression.Herein we demonstrate the quantitative nature of YS2H in predicting the affinity between two interacting proteins, particularly in the range of 100 pm to 10 μm. This feature allowed us to examine specific interactions between antigen and antibody, to identify hot spots of allosteric activation in integrins, and to isolate camelid heavy chain-only antibodies against botulinum neurotoxin as components of therapeutic agents to treat botulism (9). With the incorporation of PCA technique into the YS2H, our system may be developed into an in vivo tool to measure the kinetics of protein-protein interactions. Potential applications of YS2H include affinity maturation of antibodies, differentiation of weak to high affinity binders to the hub protein in interaction networks, and confirmation of hypothetical interacting pairs of proteins in a high throughput manner.     

Physiological Regulation of Membrane Protein Sorting Late in the Secretory Pathway of Saccharomyces cerevisiae

In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.     

Yeast Kre2 Defines a New Gene Family Encoding Probable Secretory Proteins, and Is Required for the Correct N-Glycosylation of Proteins

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.     

Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.     

Conjugation Rescue of Exocytosis Mutants in Tetrahymena thermophila Indicates the Presence of Functional Intermediates in the Regulated Secretory Pathway

ABSTRACT. Tetrahymena thermophila possesses a regulated secretory pathway in which mucin proteins are stored in dense-core granules, called mucocysts. Exocytosis-defective mutants exist that fail to secrete mucin in response to secretagogues. Four of the mutants (SB281, SB283, SB285 and SB715) appear to be blocked at different steps of the regulated secretory pathway. SB281 and SB285 accumulate mucin proteins in heterogeneous cytoplasmic organelles which have not yet been identified; SB283 makes mucocyst-like structures but they contain no immunologically identifiable 80-kDa or 50-kDa mucin proteins; and SB715 has more than normal amounts of immature and undocked mucocysts. The organelles that accumulate in exocytosis-defective mutants could be either normal intermediates in the biosynthetic pathway or aberrant structures that form as a result of the mutations. We have used conjugation rescue to analyze steps in the biogenesis of exocytosis-competent mucocysts and to identify functional intermediates. The cytoplasmic organelles that accumulate in SB281 appear to be unidentified biosynthetic intermediates, and the defect is in a cytosolic protein essential for mucocyst maturation. The organelles which accumulate in the other mutants are likely biosynthetic, but their mutations are in proteins which are labile or not free to diffuse into the mutant conjugant.     

Identification of a Developmentally Regulated Calcium-Binding Protein in Trypanosoma brucei1

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a ⁴⁵Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Palmdelphin在出生后发育的大鼠小脑中的表达模式研究

Palmdelphin是参与质膜的动态变化与细胞形态的调控的paralemmin家族新成员,与神经发育的相关性尚不明确.前期工作提示,它与调控小脑发育的一种肌动蛋白结合蛋白Mtss1(metastasis suppressor1)具有一定相关性.为了探索该基因与小脑出生后发育的相关性,利用原位杂交技术研究Palmdelphin在小脑中的时空表达,结果表明,Palmdelphin在出生后第7d大鼠小脑中有明显的表达,且分布主要集中在浦肯野神经元.半定量RT-PCR的结果进一步表明Palmdelphin的转录水平在小脑发育过程中受到调控,在出生后7d有表达高峰.这些结果显示Palmdelphin与小脑出生后神经元发育存在一定相关性.     

Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.     

Characterization and Cellular Localization of a Developmentally Regulated Rat Neural Sialidase

A developmentally regulated neural sialidase has been identified in particulate, subcellular fractions of rat brain. Enzyme activity, measured using a [3H]sialoganglioside substrate, was linear with time and had a pH optimum of 4.0-4.5. Protein linearity was only observed at low protein concentrations. This appeared to be caused by enzyme access to a lipophilic substrate, as activity was significantly stimulated by membrane-fluidizing agents. Enzyme activity was developmentally expressed in P2 pellets coincident with in vivo synaptogenesis. It was located on the synaptosome and was particularly high in myelin-containing fractions. Its cellular distribution was confined to neuronal cells and centrally derived oligodendrocytes.     

Structural Conservation of Components in the Amino Acid Sensing Branch of the TOR Pathway in Yeast and Mammals

The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR (target of rapamycin) signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins Gtr1p and Gtr2p were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 (mitogen-activated protein kinase scaffold protein 1) and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.     

The Yeast GRASP Grh1 Colocalizes with COPII and Is Dispensable for Organizing the Secretory Pathway

In mammalian cells, the ‘Golgi reassembly and stacking protein’ (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non‐essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER–Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER–Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.     

Developmentally Regulated Expression of Persyn, a Member of the Synuclein Family, in Skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.