Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.     

Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide

Cyclotriazadisulfonamide (CADA) inhibits the co‐translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)‐dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h‐region are critical for sensitivity to CADA. In particular, exchanging Gln‐15, Val‐17 or Pro‐20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N‐terminal portion of the mature protein, these residues mediate full susceptibility to the co‐translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h‐domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co‐translational translocation inhibitor.     

Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways

The tetraspanin membrane protein CD151 has been suggested to regulate cancer invasion and metastasis by initiating signaling events. The CD151-mediated signaling pathways involved in this regulation remain to be revealed. In this study, we found that stable transfection of CD151 into MelJuSo human melanoma cells lacking CD151 expression significantly increased cell motility, matrix metalloproteinase-9 (MMP-9) expression, and invasiveness. The enhancement of cell motility and MMP-9 expression by CD151 overexpression was abrogated by inhibitors and small interfering RNAs targeted to focal adhesion kinase (FAK), Src, p38 MAPK, and JNK, suggesting an essential role of these signaling components in CD151 signaling pathways. Also, CD151-induced MMP-9 expression was shown to be mediated by c-Jun binding to AP-1 sites in the MMP-9 gene promoter, indicating AP-1 activation by CD151 signaling pathways. Meanwhile, CD151 was found to be associated with alpha(3)beta(1) and alpha(6)beta(1) integrins in MelJuSo cells, and activation of associated integrins was a prerequisite for CD151-stimulated MMP-9 expression and activation of FAK, Src, p38 MAPK, JNK, and c-Jun. Furthermore, CD151 on one cell was shown to bind to neighboring cells expressing CD151, suggesting that CD151 is a homophilic interacting protein. The homophilic interactions of CD151 increased motility and MMP-9 expression of CD151-transfected MelJuSo cells, along with FAK-, Src-, p38 MAPK-, and JNK-mediated activation of c-Jun in an adhesion-dependent manner. Furthermore, C8161 melanoma cells with endogenous CD151 were also shown to respond to homophilic CD151 interactions for the induction of adhesion-dependent activation of FAK, Src, and c-Jun. These results suggest that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through FAK-Src-MAPKs pathways in human melanoma cells, leading to enhanced cell motility and MMP-9 expression.     

Increased ezrin expression and activation by CDK5 coincident with acquisition of the senescent phenotype

Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an anti-oncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes including cell cycle exit and shape changes. Here we show that senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells.     

Repression of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by binding of the virus to its primary cellular receptor, the CD4 molecule.

We have previously postulated that the binding of the human immunodeficiency virus type 1 (HIV-1) to cell surface CD4 induces signal transduction pathways that down-modulate production of progeny virions in acutely infected T cells (M. Tremblay, S. Meloche, S. Gratton, M. A. Wainberg, and R.-P. Sékaly, EMBO J. 13:774-783, 1994). To evaluate the possibility that CD4 cross-linking might indeed affect viral gene expression, we have introduced a molecular construct made of the luciferase reporter gene placed under the control of the regulatory elements of HIV-1 in several CD4-positive T-cell lines. We found that cross-linking of CD4 with defective HIV-1 particles and heat-inactivated viruses inhibits long terminal repeat-dependent luciferase expression. Experiments revealed that the gp120-CD4 interaction was necessary to repress HIV-1 long terminal repeat-dependent luciferase activity. The cytoplasmic domain of CD4 was also found to be required for this effect to occur. The virus-mediated signal transduction was shown to be mediated via p56lck-dependent and -independent pathways. These results indicate that the earliest event in the HIV-1 replicative cycle, namely, the binding of the virus to its cellular receptor, can lead to signal transduction culminating in down-modulation of viral gene expression. Thus we propose that defective viruses could regulate the pathogenesis of HIV disease as they constitute the vast majority of circulating HIV-1 particles.     

Fez1/Lzts1 a new mitotic regulator implicated in cancer development

The retinoblastoma (Rb) tumor suppressor gene product, pRb, has an established role in the implementation of cellular senescence, the state of irreversible G1 cell cycle arrest provoked by diverse oncogenic stresses. In murine cells, senescence cell cycle arrest can be reversed by subsequent inactivation of pRb, indicating that pRb is required not only for the onset of cellular senescence, but also for the maintenance of senescence program in murine cells. However, in human cells, once pRb is fully activated by p16^INK4a, senescence cell cycle arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb, suggesting that p16^INK4a/Rb-pathway activates an alternative mechanism to irreversibly block the cell cycle in human senescent cells. Here, we discuss the molecular mechanism underlying the irreversibility of senescence cell cycle arrest and its potential towards tumor suppression.     

ERM Proteins and Cdk5 in Cellular Senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-b-galactosidase (SA-b-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-b-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

Stress kinase MKK7: savior of cell cycle arrest and cellular senescence

The c-Jun N-terminal kinase (JNK/SAPK) signaling cascade controls a spectrum of cellular processes, including cell growth, differentiation, transformation, and apoptosis. We recently demonstrated that stress kinase MKK7, a direct activator of JNKs, couples stress signaling to G2/M cell cycle progression, CDC2 expression, and cellular senescence. We further explored other molecules involved in JNK pathway and found that both MKK4, another direct activator of JNK, and c-Jun, a direct substrate of JNK, have similar roles to MKK7. Here we discuss the importance of the MKK4/MKK7-JNK-c-Jun pathway linking stress and developmental signals to cell proliferation, cell cycle progression, cellular senescence, and apoptosis including recent unpublished data from our lab.     

ERM proteins and Cdk5 in cellular senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-beta-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

Sortilin Expression Is Essential for Pro-Nerve Growth Factor-Induced Apoptosis of Rat Vascular Smooth Muscle Cells

Background

Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells.

Methods and Principal Findings

Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning. In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75^NTR expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF.

Conclusions

Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.     

Targeting the retinoblastoma protein by MC007L, gene product of the molluscum contagiosum virus: detection of a novel virus-cell interaction by a member of the poxviruses

The human pathogenic poxvirus molluscum contagiosum virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxvirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a virus can perturb the cell cycle.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.     

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.     

Chaperones in cell cycle regulation and mitogenic signal transduction: a review

Chaperones/heat shock proteins (HSPs) of the HSP90 and HSP70 families show elevated levels in proliferating mammalian cells and a cell cycle-dependent expression. They transiently associate with key molecules of the cell cycle control system such as Cdk4, Wee-1, pRb, p53, p27/Kip1 and are involved in the nuclear localization of regulatory proteins. They also associate with viral oncoproteins such as SV40 super T, large T and small t antigen, polyoma large and middle S antigen and EpsteinBarr virus nuclear antigen. This association is based on a J-domain in the viral proteins and may assist their targeting to the pRb/E2F complex. Small HSPs and their state of phosphorylation and oligomerization also seem to be involved in proliferation and differentiation. Chaperones/HSPs thus play important roles within cell cycle processes. Their exact functioning, however, is still a matter of discussion. HSP90 in particular, but also HSP70 and other chaperones associate with proteins of the mitogen-activated signal cascade, particularly with the Src kinase, with tyrosine receptor kinases, with Raf and the MAP-kinase activating kinase (MEK). This apparently serves the folding and translocation of these proteins, but possibly also the formation of large immobilized complexes of signal transducing molecules (scaffolding function).     

Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1–100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 µM) decreased cell proliferation by 65% and the distribution of cells in the G₁ phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.     

Proteomics and Network Analyses Reveal Inhibition of Akt‐mTOR Signaling in CD4+ T Cells by Mycobacterium tuberculosis Mannose‐Capped Lipoarabinomannan

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4⁺ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4⁺ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4⁺ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4⁺ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4⁺ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.