A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.     

A functional link between Disrupted-In-Schizophrenia 1 and the eukaryotic translation initiation factor 3

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a candidate gene for schizophrenia. DISC1 is disrupted by a balanced t(1;11)(q42.1;q14.3) translocation segregating with schizophrenia and related psychiatric illness in a large Scottish family. Here, we show that DISC1 interacts via its globular domain with the p40 subunit of the eukaryotic translation initiation factor 3. Furthermore, we found that overexpression of DISC1 in SH-SY5Y cells induces the assembly of eIF3- and TIA-1-positive stress granules (SGs), discrete cytoplasmic granules formed in response to environmental stresses. Our findings suggest that DISC1 may function as a translational regulator and may be involved in stress response.     

Lipids are a constitutive component of cytolytic granules

Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.     

Codependent functions of RSK2 and the apoptosis-promoting factor TIA-1 in stress granule assembly and cell survival

Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the serine/threonine kinase RSK2. In stressed breast cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.     

Cell surface expression of an in vitro recombinant class II/class I major histocompatibility complex gene product

Chimeric histocompatibility genes encoding the amino-terminal (beta 1) domain of the class II Ak beta polypeptide and the carboxy-terminal (C2, transmembrane, and intracytoplasmic) domains of either the class I H-2Ld or H-2Dd molecules were stably introduced into mouse L cells. Although both were transcribed, only 5' Ak beta/3' H-2Dd transformants had significant cell membrane expression of a 30-40 kd, heterogeneous glycoprotein containing Ak beta 1 and H-2Dd (C2) serological epitopes. These transformants had a unique pattern of reactivity with monoclonal antibodies previously identified as requiring the Ak beta 1 domain for recognition of complete I-A molecules. These results allow new insight into the structural requirements for cell surface expression of proteins and provide unique cellular reagents for the dissection of humoral and cell-mediated recognition of MHC molecules.     

Stress granule assembly is mediated by prion-like aggregation of TIA-1

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.     

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.     

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.     

Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein

TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.     

Identification of FUSE-binding proteins as interacting partners of TIA proteins

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.     

Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments

Limited chymotryptic cleavage of soluble chicken gizzard desmin protofilaments allows the characterization of three structurally distinct domains. A surface-exposed very basic amino-terminal region (the headpiece) with an amino acid sequence excluding a-helical organization (7.5 kd) is separated from the perhaps globular carboxy-terminal 48 residues (the tailpiece) by a distinctly different middle domain of approximately 330 residues. This 38 kd domain is very rich in α-helix (at least 83%), and electron microscopy reveals a thin rod with a length of 500 ± 50 Å. Amino acid sequence data also show that the rod domain is interrupted by a nonhelical portion. An a-helical array is able to form a coiled-coil spanning the carboxy-terminal half of the 38 kd domain. The a-type diffraction pattern of 10 nm filaments arises from a coiled-coil conformation displayed through most but not all of the middle domain of the protofilaments.     

Murine lymphocyte and embryonal carcinoma cell surface antigens recognised by rabbit anti-murine embryonal carcinoma serum

Some recent studies have suggested that murine embryonal carcinoma (EC) cells which lack classical H-2 molecules must express some novel class-I-MHC (major histocompatibility complex) type antigens. We have investigated whether rabbit anti-EC sera may recognize such components. Molecules of 40 and 48 kd were immunoprecipitated from EC cells and lymphocytes. The possibility that these molecules may be coded for by genes in the Qa-Tla locus which contains many MHC class-I genes or pseudogenes was investigated. The 40 and 48 kd molecules were not associated with beta 2-microglobulin on EC cells, nor with each other or other molecules through S-S bonds, although they appeared to have intradisulphide structures. Peptide map analysis suggested that the lymphocyte 40 and 48 kd molecules were related to the EC cell 40 and 48 kd molecules. However, these molecules were different from H-2, Ia or Qa-2 molecules from the same mouse strain.     

Neural cell recognition molecule F11: homology with fibronectin type III and immunoglobulin type C domains

We report here the complete cDNA sequence of F11 130 kd polypeptide, a chick neural cell surface-associated glycoprotein implicated in neurite fasciculation and elongation. The predicted protein sequence of 1010 amino acids includes an amino-terminal signal peptide and a carboxy-terminal hydrophobic stretch, which is compatible with the consensus motif for covalent attachment of glycosyl-phosphatidylinositol. Accordingly, F11 lacks an intracellular domain, which is consistent with evidence obtained from protease protection experiments on isolated microsomes. In addition, the molecule comprises six domains related to the immunoglobulin domain type C and four resembling fibronectin repeat type III. Both types of repeats resemble those present in neural cell adhesion molecules L1 and N-CAM. The possible identity of F11 with the chick neural glycoprotein contactin is discussed.     

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.     

RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.     

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.     

A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a "motor" domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative "motor" domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.