67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma.

BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

Pigment Epithelium-Derived Factor and its Phosphomimetic Mutant Induce JNK-Dependent Apoptosis and P38-Mediated Migration Arrest.

BACKGROUND/AIMS: Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. METHODS: Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects. RESULTS: We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells. CONCLUSION: These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.

Oligomer formation of a tea polyphenol, EGCG, on its sensing molecule 67 kDa laminin receptor.

Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR.

Fragment-based methods for the discovery of inhibitors modulating lysyl-tRNA synthetase and laminin receptor interaction.

Lysyl-tRNA synthetase (KRS) is an enzyme that conjugates lysine to its cognate tRNAs in the process of protein synthesis. In addition to its catalytic function, KRS binds to the 67-kDa laminin receptor (LR) on the cell membrane and facilitates cell migration and metastasis. Modulation of this interaction by small-molecule inhibitors can be exploited to suppress cancer metastasis. In this study, we present fragment-based methods for the identification of inhibitors and monitoring protein-protein interactions between KRS and LR. First, we identified the amino acid residues, located on the KRS anticodon-binding domain, which interact with the C-terminal extension of the LR. One-dimensional (1D) relaxation-edited nuclear magnetic resonance spectroscopy (NMR) and competition experiments were designed and optimized to screen the fragment library. For screening using two-dimensional (2D) NMR, we identified the indicative signals in the KRS anticodon-binding domain and selected inhibitors that bind to KRS and compete with LR at the KRS-LR binding interface. These methods may offer an efficient approach for the discovery of anti-metastatic drugs.

Function of membranous lysyl-tRNA synthetase and its implication for tumorigenesis.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that conjugate specific amino acids to their cognate tRNAs for protein synthesis. Besides their catalytic activity, recent studies have uncovered many additional functions of these enzymes through their interactions with diverse cellular factors. Among human ARSs, cytosolic lysyl-tRNA synthetase (KRS) is often highly expressed in cancer cells and tissues, and facilitates cancer cell migration and invasion through the interaction with the 67kDa laminin receptor on the plasma membrane. Specific modulation of this interaction by small molecule inhibitors has revealed a new way to control metastasis. Here, we summarize the pro-metastatic functions of KRS and their patho-physiological implications.

The Transition of the 37-Kda Laminin Receptor (Rpsa) to Higher Molecular Weight Species: Sumoylation or Artifact?

The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

Discovery of new small molecules inhibiting 67 kDa laminin receptor interaction with laminin and cancer cell invasion.

The 67 kDa laminin receptor (67LR) is a non-integrin receptor for laminin (LM) that derives from a 37 kDa precursor (37LRP). 67LR expression is increased in neoplastic cells and correlates with an enhanced invasive and metastatic potential. We used structure-based virtual screening (SB-VS) to search for 67LR inhibitory small molecules, by focusing on a 37LRP sequence, the peptide G, able to specifically bind LM. Forty-six compounds were identified and tested on HEK-293 cells transfected with 37LRP/67LR (LR-293 cells). One compound, NSC47924, selectively inhibited LR-293 cell adhesion to LM with IC50 and Ki values of 19.35 and 2.45 µmol/L. NSC47924 engaged residues W176 and L173 of peptide G, critical for specific LM binding. Indeed, NSC47924 inhibited in vitro binding of recombinant 37LRP to both LM and its YIGSR fragment. NSC47924 also impaired LR-293 cell migration to LM and cell invasion. A subsequent hierarchical similarity search with NSC47924 led to the identification of additional four compounds inhibiting LR-293 cell binding to LM: NSC47923, NSC48478, NSC48861, and NSC48869, with IC50 values of 1.99, 1.76, 3.4, and 4.0 µmol/L, respectively, and able to block in vitro cancer cell invasion. These compounds are promising scaffolds for future drug design and discovery efforts in cancer progression.

67-kDa Laminin receptor contributes to hypoxia-induced migration and invasion of trophoblast-like cells by mediating matrix metalloproteinase-9.

Insufficient trophoblast invasion often occurs in patients experiencing preeclampsia. The 67-kDa laminin receptor (LR1) is a multifunctional protein that binds to laminin and interacts with the extracellular matrix. We recently demonstrated that LR1 is implicated in trophoblast migration and invasion. However, whether LR1 is involved in hypoxia-mediated trophoblastic invasion remains unclear and requires further investigation. This study demonstrates that two trophoblast-like cell lines (JEG3 and BeWo cells) cultured at 3% oxygen exerted enhanced migratory and invasive capabilities as compared with their counterparts exposed to 20% oxygen. LR1 expression was increased in hypoxic JEG3 cells but decreased after transfection with hypoxia-inducible factor 1 alpha (HIF-1α) specific siRNA. Moreover, shRNA targeting LR1 mRNA significantly inhibited hypoxia-induced increase in matrix metalloproteinase (MMP)-9 activity in JEG3 cells. Forced overexpression of LR1 augmented JEG3 cell migration and invasion, and enhanced MMP-9 expression and activity. Additionally, the blockade of the MMP-9 effect with its neutralizing antibody reduced LR1 elevation-promoted trophoblastic invasion. In summary, this study demonstrates that LR1 contributes to hypoxia-induced migration and invasion of trophoblast cells at least partly by mediating MMP-9 in vitro.

Characterization of the interaction between lysyl-tRNA synthetase and laminin receptor by NMR.

Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS-37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS-LR interaction on the cell surface.

High resolution imaging study of interactions between the 37 kDa/67 kDa laminin receptor and APP, beta-secretase and gamma-secretase in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia affecting the elderly. Neurodegeneration is caused by the amyloid beta (Aß) peptide which is generated from the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by the ß- and γ- secretases. Previous reports revealed that the 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in APP processing, however, the exact mechanism by which this occurs remains largely unclear. This study sought to assess whether LRP/LR interacted with APP, ß- or γ-secretase. Detailed confocal microscopy revealed that LRP/LR showed a strong co-localisation with APP, ß- and γ-secretase, respectively, at various sub-cellular locations. Superresolution Structured Illumination Microscopy (SR-SIM) showed that interactions were unlikely between LRP/LR and APP and ß-secretase, respectively, while there was strong co-localisation between LRP/LR and γ-secretase at this 80 nm resolution. FRET was further employed to assess the possibility of protein-protein interactions and only an interaction between LRP/LR and γ-secretase was found. FLAG co-immunoprecipitation confirmed these findings as LRP/LR co-immunoprecipitated with γ-secretase, but failed to do so with APP. These findings indicate that LRP/LR exerts its influence on Aß shedding via a direct interaction with the γ-secretase and possibly an indirect interaction with the ß-secretase.

Green tea catechins potentiate the neuritogenic action of brain-derived neurotrophic factor: role of 67-kDa laminin receptor and hydrogen peroxide.

Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 µg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 µM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H2O2 in the potentiation. Consistently, exogenous sublethal concentrations of H2O2, added as a bolus dose (5 µM) or more effectively through a steady-state generation (1 µM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67 LR and H2O2, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.

Parkin induces upregulation of 40S ribosomal protein SA and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells.

Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.

67-kDa laminin receptor increases cGMP to induce cancer-selective apoptosis.

The 67-kDa laminin receptor (67LR) is a laminin-binding protein overexpressed in various types of cancer, including bile duct carcinoma, colorectal carcinoma, cervical cancer, and breast carcinoma. 67LR plays a vital role in growth and metastasis of tumor cells and resistance to chemotherapy. Here, we show that 67LR functions as a cancer-specific death receptor. In this cell death receptor pathway, cGMP initiated cancer-specific cell death by activating the PKCδ/acid sphingomyelinase (PKCδ/ASM) pathway. Furthermore, upregulation of cGMP was a rate-determining process of 67LR-dependent cell death induced by the green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG), a natural ligand of 67LR. We found that phosphodiesterase 5 (PDE5), a negative regulator of cGMP, was abnormally expressed in multiple cancers and attenuated 67LR-mediated cell death. Vardenafil, a PDE5 inhibitor that is used to treat erectile dysfunction, significantly potentiated the EGCG-activated 67LR-dependent apoptosis without affecting normal cells and prolonged the survival time in a mouse xenograft model. These results suggest that PDE5 inhibitors could be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death.

67 kDa laminin receptor: structure, function and role in cancer and infection.

The 67 kDa high affinity laminin receptor (67LR) is a non integrin cell surface receptor for the extracellular matrix whose expression is increased in neoplastic cells and directly correlates with an enhanced invasive and metastatic potential. 67LR derives from homo- or hetero-dimerization of a 37 kDa cytosolic precursor (37LRP), by fatty acid acylation. Interestingly, 37LRP is a multifunctional protein involved in the translational machinery and has also been found in the nucleus, where it is tightly associated with nuclear structures. Acting as a receptor for laminin is not the only function of this protein; indeed, 67LR also acts as a receptor for viruses, such as Sindbis virus and Dengue virus, and is involved in the internalization of the prion protein. Here, we review the current understanding of the structure and function of this molecule, highlighting its role in cancer and infection diseases.

Comprehensive proteomic analysis of nonintegrin laminin receptor interacting proteins.

Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion proteins, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from Mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through coimmunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/metabolism, protein processing, cytoskeleton/cell anchorage, DNA/chromatin, and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor and provides an essential stepping stone to a better mechanistic understanding of this protein's diverse functions.

Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG.

(-)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O(2) levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O(2) levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O(2). In summary, 67LR expression is highly sensitive to O(2) partial pressure, and the activity of EGCG can be regulated in cancer cells by O(2) partial pressure.

Green tea polyphenol EGCG sensing motif on the 67-kDa laminin receptor.

BACKGROUND: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. CONCLUSIONS/SIGNIFICANCE: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.

Multiple folding states and disorder of ribosomal protein SA, a membrane receptor for laminin, anticarcinogens, and pathogens.

The human ribosomal protein SA (RPSA) is a multilocus protein, present in most cellular compartments. It is a multifunctional protein, which belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogenic microorganisms, toxins, and the anticarcinogen epigallocatechin gallate. It contributes to the crossing of the blood-brain barrier by neurotropic viruses and bacteria and is used as a biomarker of metastasis. RPSA includes an N-terminal domain, which is homologous to the prokaryotic ribosomal proteins S2, and a C-terminal extension, which is conserved in vertebrates. The structure of its N-domain has been determined from crystals grown at 17 °C. The structure of its C-domain remains unknown. We produced in Escherichia coli and purified the full-length RPSA and its N- and C-domains. We characterized the folding states of these recombinant proteins mainly by methods of fluorescence and circular dichroism spectrometry, in association with quantitative analyses of their unfolding equilibria, induced with heat or urea. The necessary equations were derived from first principles. The results showed that the N-domain unfolded according to a three-state equilibrium. The monomeric intermediate was predominant at the body temperature of 37 °C. It also existed in the full-length RPSA and bound ANS, a small fluorescent molecule. The C-domain was in an intrinsically disordered state. The recombinant N- and C-domains weakly interacted together. These results indicated a high plasticity of RPSA, which could be important for its multiple cellular localizations and functional interactions.

Conformational switch of a flexible loop in human laminin receptor determines laminin-1 interaction.

The 37/67-kDa human laminin receptor(LamR) is a cell surface protein that interacts with molecules located in the extra-cellular matrix. In particular, interactions between LamR and laminins play a major role in mediating changes in the cellular environment that affect cell adhesion, neurite outgrowth, tumor growth and metastasis. The exact interaction mode of laminin-1 and LamR is not fully understood. Laminin-1 is thought to bind to LamR through interaction with the so-called peptide G (residues 161­180) and the C-terminal helix (residues 205­229). Here we performed 100-ns atomistic force field based molecular dynamics simulations to explore the structure and dynamics of LamR related to laminin-1 interactions. Our main finding is that loop 188­197 in the C-terminal region is highly flexible. It undergoes a major change resulting in a conformational switch that partially solvent exposes the R180 residue in the final part of the G peptide. So, R180 could contribute to laminin-1 binding. Projection of the simulations along the first two principal components also confirms the importance of this conformational switch in the LamR. This may be a basic prerequisite to clarify the key structural determinants of the interaction of LamR with laminin-1.