Expression of heparin/heparan sulfate interacting protein/ribosomal protein l29 during the estrous cycle and early pregnancy in the mouse

Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.     

Repression of HIP/RPL29 expression induces differentiation in colon cancer cells

We had previously shown that the expression of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) was upregulated in colon cancer tissues. The present study investigated the role of HIP/RPL29 in differentiation in colon cancer cells. Inducing cellular differentiation in HT-29 cells by both sodium butyrate and glucose deprivation resulted in a significant downregulation of HIP/RPL29 expression. The beta-catenin/Tcf-4 pathway is the most important pathway controlling the switch between cellular differentiation and proliferation in intestinal epithelial cells. Inducing differentiation by dominant-negative inhibition of the beta-catenin/Tcf-4 complexes in LS174T cells also resulted in downregulation of HIP/RPL29. To determine whether a lower expression of HIP/RPL29 could induce differentiation in cancer cells, small interfering RNA (siRNA) targeting HIP/RPL29 was transfected into LS174T cells. The resultant knockdown of HIP/RPL29 expression induced cellular differentiation, as shown by the increased expression of two known markers of differentiation in LS174T cells, galectin-4 and mucin-2. In addition, the differentiation process induced by repression of HIP/RPL29 expression was accompanied by the upregulation of p21 and p53. In conclusion, HIP/RPL29 plays a role in the cellular differentiation process in colon cancer cells. The differentiation process is at least partially mediated by the upregulation of p21 and p53 pathways.     

Comparative mapping of RPL3, a gene overexpressed in multiple obesity models.

The ribosomal protein 3 gene is differentially expressed in hypothalamus and brown adipose tissue between mouse lines divergently selected for heat loss, and in skeletal muscle of the ob/ob mouse model. Unfortunately, multiple Rpl3-processed pseudogenes have hampered mapping of the functional gene copy in mammalian species. Using PCR amplification with intronic primer binding, we have mapped Rpl3 to MMU15, and have also localized RPL3 to BTA5 in cattle. Comparative mapping implicates a previously mapped copy of RPL3 on HSA22 as the functional copy of human RPL3, while predictive mapping places the porcine homologue on SSC5.     

HIP/RPL29 antagonizes VEGF and FGF2 stimulated angiogenesis by interfering with HS-dependent responses

HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29's ability to modulate actions of HS-binding growth factors (HBGFs) in angiogenesis. Between 1 and 2.5 microg/ml (ca. 60-150 nM), HIP/RPL29 inhibited HBGF-stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 microg/ml). At this concentration, HIP/RPL29 had no effect on HBGF-stimulated MAPK phosphorylation or VEGF-stimulated receptor-2 phosphorylation at site Y-996. Partial inhibition occurred at VEGF receptor-2 site Y951, associated with cell migration. HBGF displacement from HS-bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 microg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE-dependent release of HS-bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 microg/ml and by 77% at 40 microg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 microg/ml (ca. 2.5 microM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors.     

Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell growth in the cancerous state, and the modulation of blood coagulation. These activities are consistent with the proposed function of HIP/L29 as a heparin/heparan sulfate (Hp/HS) binding growth factor that has a preference for anticoagulantly active Hp/HS. Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion. However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the ability to bind Hp, suggesting that there may be additional Hp/HS binding sites outside of the HIP peptide-1 domain. To test this hypothesis, a systematic study of the domains within human and murine HIP/L29 responsible for Hp/HS binding activity was undertaken. Using deletion mutants, proteolytic fragments, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins. Furthermore, a conformational change is induced in human HIP/L29 upon Hp binding as detected by circular dichroism spectroscopy. These studies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.     

RPL29 codes for a non-essential protein of the 60S ribosomal subunit in Saccharomyces cerevisiae and exhibits synthetic lethality with mutations in genes for proteins required for subunit coupling

RPL29 (YFR032c-a) is a non-essential gene that codes for a 60S ribosomal subunit protein in Saccharomyces cerevisiae. Deletion of RPL29 leads to a moderate accumulation of half-mer polysomes with little or no change in the amounts of free 60S subunits. In vitro translation and the growth rate are also delayed in the Deltarpl29 strain. Such a phenotype is characteristic of mutants defective in 60S to 40S subunit joining. The Deltarpl29 strain exhibits synthetic lethality with mutations in RPL10, the gene encoding an essential 60S ribosomal subunit protein that is required for 60S to 40S subunit joining. The Deltarpl29 strain also exhibits synthetic lethality with RSA1, a gene encoding a nucleoplasmic protein required for the loading of Rpl10p onto the 60S subunit. Over-expression of RPL10 suppresses the half-mer phenotype of the Deltarpl29 strain, but does not correct the growth defect of the deletion strain. We conclude that absence of Rpl29p impairs proper assembly of proteins onto the 60S subunit and that this retards subunit joining and additionally retards protein synthesis subsequent to subunit joining.     

Low levels of human HIP14 are sufficient to rescue neuropathological, behavioural, and enzymatic defects due to loss of murine HIP14 in Hip14-/- mice

Huntingtin Interacting Protein 14 (HIP14) is a palmitoyl acyl transferase (PAT) that was first identified due to altered interaction with mutant huntingtin, the protein responsible for Huntington Disease (HD). HIP14 palmitoylates a specific set of neuronal substrates critical at the synapse, and downregulation of HIP14 by siRNA in vitro results in increased cell death in neurons. We previously reported that mice lacking murine Hip14 (Hip14-/-) share features of HD. In the current study, we have generated human HIP14 BAC transgenic mice and crossed them to the Hip14-/- model in order to confirm that the defects seen in Hip14-/- mice are in fact due to loss of Hip14. In addition, we sought to determine whether human HIP14 can provide functional compensation for loss of murine Hip14. We demonstrate that despite a relative low level of expression, as assessed via Western blot, BAC-derived human HIP14 compensates for deficits in neuropathology, behavior, and PAT enzyme function seen in the Hip14-/- model. Our findings yield important insights into HIP14 function in vivo.     

Differential subcellular localization of ribosomal protein L7 paralogs in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In Saccharomyces cerevisiae, ribosomal protein L7, one of the ∼46 ribosomal proteins of the 60S subunit, is encoded by paralogous RPL7A and RPL7B genes. The amino acid sequence identity between Rpl7a and Rpl7b is 97 percent; they differ by only 5 amino acid residues. Interestingly, despite the high sequence homology, Rpl7b is detected in both the cytoplasm and the nucleolus, whereas Rpl7a is detected exclusively in the cytoplasm. A site-directed mutagenesis experiment revealed that the change in the amino acid sequence of Rpl7b does not influence its sub-cellular localization. In addition, introns of RPL7A and RPL7B did not affect the subcellular localization of Rpl7a and Rpl7b. Remarkably, Rpl7b was detected exclusively in the cytoplasm in rpl7a knockout mutant, and overexpression of Rpl7a resulted in its accumulation in the nucleolus, indicating that the subcellular localization of Rpl7a and Rpl7b is influenced by the intracellular level of Rpl7a. Rpl7b showed a wide range of localization patterns, from exclusively cytoplasmic to exclusively nucleolar, in knock-out mutants for some rRNA-processing factors, nuclear pore proteins, and large ribosomal subunit assembly factors. Rpl7a, however, was detected exclusively in the cytoplasm in these mutants. Taken together, these results suggest that although Rpl7a and Rpl7b are paralogous and functionally replaceable with each other, their precise physiological roles may not be identical.     

The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog,Rpl22l1

Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22^−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22^−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.     

High expression of the human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene in the mammary gland of lactating transgenic mice. Secretion into the milk and purification of the HIP/PAP lectin.

The human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene was previously identified because of its increased expression in primary liver cancers and during the acute phase of pancreatitis. In normal tissues, HIP/PAP is expressed both in endocrine and exocrine cells of the intestine and pancreas. HIP/PAP is a lactose binding C-type lectin which acts as an adhesion molecule for rat hepatocytes. The aim of the work was to study the HIP/PAP secretory pathway and to produce high levels of HIP/PAP in the milk of lactating transgenic mice. In view of its lactose C-type lectin properties, we have studied the consequences of the expression of HIP/PAP on mammary epithelial cells. In homozygous mice, production reached 11.2 mg.mL-1 of milk. High levels of soluble and pure HIP/PAP (18.6 mg) were purified from 29 mL of milk. The purified protein was sequenced and the N-terminal amino acid of the mature HIP/PAP was identified as Glu27, thus localizing the site of cleavage of the signal peptide. The HIP/PAP transgene was only expressed in the mammary gland of lactating transgenic mice. HIP/PAP was detected by immunofluorescence in the whole gland, but labelling was heterogeneous between alveolar clusters, with strongly positive sparse cells. Using immuno electron microscopy, HIP/PAP was observed in all the compartments of the secretory pathway within the mammary epithelial cells. We provide evidence that HIP/PAP is secreted through the Golgi pathway. However, the number of distended Golgi saccules was increased when compared to that found in wild-type mouse mammary cells. These modifications could be related to HIP/PAP C-type lectin specific properties.     

Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Δrpl31aΔrpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Δrpl31aΔrpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Δrpl31aΔrpl31bΔzuo1 did not exceed that of Δrpl31aΔrpl31b or Δzuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Δrpl31aΔrpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.