Cloning of cDNA for regenectin, a humoral C-type lectin of Periplaneta americana, and expression of the regenectin gene during leg regeneration

We isolated cDNA for regenectin, a C-type lectin of the American cockroach (Periplaneta americana), and analysed expression of the regenectin gene in the regenerating legs. Regenectin was found to be a member of the Periplaneta lectin-related protein family. We found that the regenectin gene was expressed specifically in the epidermal cells of the newly formed regenerating legs. Together with our previous results, these results suggest that regenectin is synthesized by epidermal cells, secreted into the regenerating leg saccule, and assembles around myoblasts to form leg muscle fibers in situ.     

Purification and localization of p10, a novel protein that increases in nymphal regenerating legs of Periplaneta americana (American cockroach).

Content of a protein with a molecular mass of 10 kDa (p10) was found to increase significantly in regenerating legs of the American cockroach (Periplaneta americana). This protein was purified to homogeneity from a homogenate of regenerating legs. Partial amino acid sequencing indicated that p10 is a novel protein. Immunoblotting showed that its content in regenerating legs was 30 times that in normal legs, and decreased significantly when leg regeneration was complete. An immunofluorescence study revealed that p10 localizes exclusively on the external side of newly formed epidermis of regenerating legs.     

Independent regulation of Rap1 and mitogen-activated protein kinase by the alpha chain of Go

Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.     

Radioautographic characterization of successive compartments along the rough endoplasmic reticulum-Golgi pathway of collagen precursors in foot pad fibroblasts of [3H]proline-injected rats

Young rats given an intravenous injection of [3H]proline were killed at successive times from 4 to 80 min later. Fibroblasts from the front foot pad were radioautographed ; silver grains were counted over several of the organelles and the results were expressed as percent radiolabel per unit volume. These percentages reached a peak over rough endoplasmic reticulum cisternae at 4 min, intermediate vesicles and tubules at 10 min, spherical distensions of cis-side Golgi saccules at 20 min, cylindrical distensions of trans-side saccules between 40 and 60 min, and secretory granules at 60 min. It is proposed that the succession of peaks corresponds to the migration pathway of collagen precursor proteins within fibroblasts; that is, the proteins synthesized in rough endoplasmic reticulum are delivered by intermediate vesicles and/or tubules to the spherical distensions of cis-side saccules, somehow pass from there to the cylindrical distensions of trans-side saccules and, finally, are carried by secretory granules to the extracellular space.     

Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein.

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.     

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.     

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.     

The receptor-associated protein (RAP) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.     

Rutin treats myocardial damage caused by pirarubicin via regulating miR‐22‐5p‐regulated RAP1/ERK signaling pathway

Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP‐induced myocardial injury and identified miR‐22‐5p and the RAP1‐member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR‐22‐5p (P < .01) and increases the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). RUT significantly improved THP‐induced myocardial damage (P < .01), increased the expression of miR‐22‐5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR‐22‐5p. miR‐22‐5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP‐induced myocardial damage, which may be achieved by upregulating miR‐22‐5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway.     

Synthesis, cloning and expression of recombinant aprotinin

Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E. coli. The synthetic gene is characterized by the presence of certain restriction sites. These restriction sites are unique within the used cloning system. Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments. Using this method the variant [Glu52]aprotinin was obtained starting from the aprotinin gene. Both genes were successfully expressed in E. coli as fusion proteins with beta-galactosidase using vector pUR 278. No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8). [Glu52]aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein. This recombinant [Glu52]aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin.