Biomedical subjects

Elena Kalinina

Publications and source records attributed to Elena Kalinina.

8 recordsLinked to original sources

PubMed2006

Drosophila S2 cells produce multiple forms of carboxypeptidase D with different intracellular distributions.

Carboxypeptidase D (CPD) functions in the processing of proteins that transit the secretory pathway, and is present in all vertebrates examined as well as Drosophila. Several forms of CPD mRNA were previously found in Drosophila that resulted from differential splicing of the gene. In the present study, Northern blot, reverse transcriptase PCR, and Western blot analysis showed that each splice variant occurs in a single cell type, the Drosophila-derived Schneider 2 (S2) cell line. The short forms containing a single carboxypeptidase domain were secreted from the S2 cells while the long forms containing three carboxypeptidase domains, a transmembrane domain, and one of two different cytosolic tails were retained in the cell. To investigate the role of the two different C-terminal tail sequences (tail-1 and tail-2) that result from the differential splicing within exon 8, constructs containing a reporter protein (albumin) attached to the transmembrane domain and tail-1 or tail-2 of CPD were expressed in S2 cells and a mouse pituitary cell line (AtT20 cells). Immunofluorescence analysis revealed different intracellular distributions of the two constructs, with the tail-2 construct showing considerable overlap with a Golgi marker. The two C-terminal tail sequences also resulted in different internalization efficiencies from the cell surface in both cell lines. Interestingly, the distribution and routing of the tail-2 form of Drosophila CPD in the AtT20 cells are similar to the previously characterized endogenous mouse CPD protein, indicating that the elements for this trafficking have been conserved between Drosophila and mammals.

Alternative Splicing↗

PubMed2006

Carboxypeptidase E in the mouse placenta.

Carboxypeptidase E (CPE) has important functions in processing of endocrine pro-peptides, such as pro-insulin, pro-opiomelanocortin, or pro-gonadotropin-releasing hormone, as evidenced by the hyper-pro-insulinemia, obesity, and sterility of Cpe mutant mice. Down-regulation of Cpe in enlarged placentas of interspecific hybrid (interspecies hybrid placental dysplasia (IHPD)) and cloned mice suggested that reduced CPE enzyme and receptor activity could underlie abnormal placental phenotypes. In this study, we have explored the role of Cpe in murine placentation by determining its expression at various stages of gestation, and by phenotypic analysis of Cpe mutant placentas. Our results show that Cpe and Carboxypeptidase D (Cpd), another carboxypeptidase with a very similar function, are strictly co-localized in the mouse placenta from late mid-gestation to term. We also show that absence of CPE causes a sporadic but striking placental phenotype characterized by an increase in giant and glycogen cell numbers and giant cell hypertrophy. Microarray-based transcriptional profiling of Cpe mutant placentas identified only a very small number of genes with altered expression, including Dtprp, which belongs to the prolactin gene family. Concordant deregulation of Cpe and Cpd in abnormal placentas of interspecies hybrids before the onset of IHPD phenotype and recapitulation of some phenotypes of IHPD hyperplastic placentas in Cpe mutant placentas suggests that these two genes are causally involved in IHPD and may function as speciation genes in the genus Mus.

Animals↗

PubMed2005

Identification and distribution of mouse carboxypeptidase A-6.

Carboxypeptidase A-6 (CPA6) was recently discovered in the human genome. To gain information regarding the potential function of this novel protein, the mouse homolog of CPA6 was identified using a combination of bioinformatics and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, homologs in rat, chicken, and frog were identified using a bioinformatics approach. The distribution of CPA6 mRNA in mouse tissues was examined using RT-PCR and in situ hybridization. A strong RT-PCR signal is detectable in olfactory bulb, and much lower levels are present in other regions such as the cerebral cortex, hippocampus, hypothalamus, striatum, and medulla. In peripheral tissues, a moderate RT-PCR signal is present in epididymis, and low levels are detectable in colon and spleen. The high level of CPA6 in adult mouse brain olfactory bulb was confirmed by in situ hybridization. Lower levels of CPA6 mRNA were found to be present in the cingulate cortex, lateral septum, pontine nucleus, and inferior olivary nucleus of the hindbrain. Within the olfactory bulb, CPA6 mRNA is enriched in the mitral and granular layer. A lower level of CPA6 mRNA is present in the internal and external plexiform layers, and no signal is detectable in the olfactory nerve layer. The distribution was also examined in whole embryos at embryonic day 14.5 and CPA6 mRNA was found to be enriched in eye, ear, osteoblasts, stomach, skin, dorsal root ganglia, and throughout the CNS. The presence of CPA6 mRNA in the rectus muscle layer of the eye at embryonic day 14.5 is consistent with the observation that the CPA6 gene is disrupted in a patient with Duane syndrome, a congenital eye defect. Taken together, the distribution of CPA6 suggests a specific role in a limited number of tissues, and it is possible that this role involves an aspect of cell migration.

Animals↗

PubMed2004

Peptidomics of Cpe fat/fat mouse hypothalamus: effect of food deprivation and exercise on peptide levels.

Carboxypeptidase E is a major enzyme in the biosynthesis of numerous neuroendocrine peptides. Previously, we developed a technique for the isolation of neuropeptide-processing intermediates from mice that lack carboxypeptidase E activity (Cpe fat/fat mice) due to a naturally occurring point mutation. In the present study, we used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic peptides in Cpe fat/fat mice in two different paradigms that each cause an approximately 10% decrease in body mass. One paradigm involved a 2-day fast under normal sedentary conditions (i.e. standard mouse cages); the other involved giving mice access to an exercise wheel for 4 weeks with free access to food. Approximately 50 peptides were detected in both studies, and over 80 peptides were detected in at least one of the two studies. Twenty-eight peptides were increased >50% by food deprivation, and some of these were increased by 2- to 3-fold. In contrast, only three peptides were increased >50% in the group with exercise wheels, and many peptides showed a slight 15-30% decrease upon exercise. Approximately one-half of the peptides detected in both studies were identified by tandem mass spectrometry. Peptides found to be elevated by food deprivation but not exercise included a number of fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin B, and pro-SAAS. Taken together, the differential regulation of these peptides in the two paradigms suggests that the regulation is not due to the lower body weight but to the manner in which the paradigms achieved this lower body weight.

Amino Acid Sequence↗

PubMed2004

Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells.

In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.

Albumins↗

PubMed2004

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.

Amino Acid Sequence↗

PubMed2003

Neuropeptide-processing carboxypeptidases.

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this "traditional" pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named "CPA-5," has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites.

Animals↗

PubMed2002

Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking.

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.

Adaptor Protein Complex alpha Subunits↗