Barsanjit Mazumder

Ceruloplasmin and cardiovascular disease

Transition metal ion-mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His(426), is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.

Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-κB-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth

ABSTRACT The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-κB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

Transcript-Selective Translational Silencing by Gamma Interferon Is Directed by a Novel Structural Element in the Ceruloplasmin mRNA 3′ Untranslated Region

ABSTRACT Transcript-selective translational control of eukaryotic gene expression is often directed by a structural element in the 3′ untranslated region (3′-UTR) of the mRNA. In the case of ceruloplasmin (Cp), induced synthesis of the protein by gamma interferon (IFN-γ) in U937 monocytic cells is halted by a delayed translational silencing mechanism requiring the binding of a cytosolic inhibitor to the Cp 3′-UTR. Silencing requires the essential elements of mRNA circularization, i.e., eukaryotic initiation factor 4G, poly(A)-binding protein, and poly(A) tail. We here determined the minimal silencing element in the Cp 3′-UTR by progressive deletions from both termini. A minimal, 29-nucleotide (nt) element was determined by gel shift assay to be sufficient for maximal binding of the IFN-γ-activated inhibitor of translation (GAIT), an as-yet-unidentified protein or complex. The interaction was shown to be functional by an in vitro translation assay in which the GAIT element was used as a decoy to overcome translational silencing. Mutation analysis showed that the GAIT element contained a 5-nt terminal loop, a weak 3-bp helix, an asymmetric internal bulge, and a proximal 6-bp helical stem. Two invariant loop residues essential for binding activity were identified. Ligation of the GAIT element immediately downstream of a luciferase reporter conferred the translational silencing response to the heterologous transcript in vitro and in vivo; a construct containing a nonbinding, mutated GAIT element was ineffective. Translational silencing of Cp, and possibly other transcripts, mediated by the GAIT element may contribute to the resolution of the local inflammatory response following cytokine activation of macrophages.

DELAYED TRANSLATIONAL SILENCING OF CERULOPLASMIN TRANSCRIPT IN GAMMA INTERFERON-ACTIVATED U937 MONOCYTIC CELLS : ROLE OF THE 3' UNTRANSLATED REGION

ABSTRACT Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-γ) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-γ for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-γ treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-γ for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3′ untranslated region (3′-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3′-UTR added as a “decoy” and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3′-UTR indicated an internal 100-nucleotide region of the Cp 3′-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

ABSTRACT Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-γ) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3′ untranslated region (UTR). To investigate how protein interactions at the Cp 3′-UTR inhibit translation initiation at the distant 5′ end, we considered the “closed-loop” model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-γ-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3′-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3′-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

Interleukin-1 Receptor-associated Kinase 2 Is Critical for Lipopolysaccharide-mediated Post-transcriptional Control

IRAK2, a member of the interleukin-1 receptor-associated kinase (IRAK) family, has been implicated in Toll-like receptor (TLR)-mediated signaling. We generated IRAK2-deficient mice to examine its function in detail. These mice are resistant to lipopolysaccharide-induced septic shock, because of impaired TLR4-mediated induction of pro-inflammatory cytokines and chemokines. Although IRAK2 deficiency did not affect TLR4-mediated NFκB activation, a reduction of lipopolysaccharide (LPS)-mediated mRNA stabilization contributed to the reduced cytokine and chemokine production observed in bone marrow-derived macrophages from IRAK2-deficient mice. Furthermore, the ratios of LPS-induced cytokine and chemokine mRNAs in translation-active (polysomal) versus translation-inactive (free ribosomes) pools were reduced in IRAK2-deficient macrophages compared with wild type macrophages. Importantly, LPS-induced phosphorylation of MKK3/6, MNK1, and eIF4E was significantly reduced in IRAK2-deficient macrophages compared with wild type macrophages. Moreover, LPS stimulation induced an interaction of IRAK2 with TRAF6, MKK3/6, and MK2, implicating a critical role for mitogen-activated protein kinase signaling in LPS-induced IRAK2-mediated post-transcriptional control. These results reveal that IRAK2 is required for LPS-mediated post-transcriptional control of cytokine and chemokine expression, which plays an essential role in TLR4-induced septic shock.

A new framework for understanding IRES-mediated translation.

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m(7)G-assisted internal initiation).

Genome-Wide Polysome Profiling Reveals an Inflammation-Responsive Posttranscriptional Operon in Gamma Interferon-Activated Monocytes

ABSTRACT We previously showed that ribosomal protein L13a is required for translational silencing of gamma interferon (IFN-γ)-induced ceruloplasmin (Cp) synthesis in monocytes. This silencing also requires the presence of the GAIT (IFN-gamma activated inhibitor of translation) element in the 3′ untranslated region (UTR) of Cp mRNA. Considering that Cp is an inflammatory protein, we hypothesized that this mechanism may have evolved to silence a family of proinflammatory proteins, of which Cp is just one member. To identify the other mRNAs that are targets for this silencing, we performed a genome-wide analysis of the polysome-profiled mRNAs by using an Affymetrix GeneChip and an inflammation-responsive gene array. A cluster of mRNAs encoding different chemokines and their receptors was identified as common hits in the two approaches and validated by real-time PCR. In silico predicted GAIT hairpins in the 3′ UTRs of the target mRNAs were confirmed as functional cis-acting elements for translational silencing by luciferase reporter assays. Consistent with Cp, the newly identified target mRNAs also required L13a for silencing. Our studies have identified a new inflammation-responsive posttranscriptional operon that can be regulated directly at the level of translation in IFN-γ-activated monocytes. This regulation of a cohort of mRNAs encoding inflammatory proteins may be important to resolve inflammation.

Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites

ABSTRACT Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.

Translation Control: A Multifaceted Regulator of Inflammatory Response

A robust innate immune response is essential to the protection of all vertebrates from infection, but it often comes with the price tag of acute inflammation. If unchecked, a runaway inflammatory response can cause significant tissue damage, resulting in myriad disorders, such as dermatitis, toxic shock, cardiovascular disease, acute pelvic and arthritic inflammatory diseases, and various infections. To prevent such pathologies, cells have evolved mechanisms to rapidly and specifically shut off these beneficial inflammatory activities before they become detrimental. Our review of recent literature, including our own work, reveals that the most dominant and common mechanism is translational silencing, in which specific regulatory proteins or complexes are recruited to cis-acting RNA structures in the untranslated regions of single or multiple mRNAs that code for the inflammatory protein(s). Enhancement of the silencing function may constitute a novel pharmacological approach to prevent immunity-related inflammation.