Signal Sequence Binding Research Articles

Identification of an immune-related six-long noncoding RNA signature as a novel prognosis biomarker for adenocarcinoma of lung.

Background: Lung adenocarcinoma (LUAD) is a heterogeneous disease with high mortality. Close attention has been paid to immunotherapy in LUAD treatment. However, immunotherapy has produced different therapeutic effects because of immune heterogeneity. Long noncoding RNAs (lncRNAs) are survival prognostic indicators with functions in the immune process. The present study was designed to examine the predictive power of immune-related lncRNAs in LUAD prognosis and investigated potential molecular mechanisms.Methods: Transcriptome profiling and LUAD sample clinical information were retrieved from online database. The immune-related lncRNAs signature was identified by Cox regression. Survival analysis was used to verify the validity of the prognosis model. Then, possible biological functions were predicted and the abundance of infiltrating immune cells in LUAD samples were further analyzed.Results: An immune-associated lncRNAs signature was established by combining six lncRNAs. Patients with LUAD were stratified into high- and low-risk groups using the six lncRNAs signature. Patients in different risk levels had significantly different prognoses (P<0.001), and the immune-associated lncRNAs signature was identified as an independent prognostic factor for LUAD. The functions of the lncRNA signature were confirmed as ubiquitin mediated proteolysis and signal sequence binding. The lncRNA signature negatively correlates with B-cell immune infiltration.Conclusion: A reliable immune-related lncRNAs prognosis model for LUAD was identified. lncRNAs played a vital role in the tumor immune process and were associated with the LUAD prognosis. Research of lncRNAs in B-cell immune infiltration could provide new insight into the immunotherapy of LUAD.

Voltage Sensitivity of the Bacterial Protein Translocation Channel

The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes. The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.1. Saparov, S.M., Erlandson, K., Cannon, K., Schaletzky, J., Schulman, S., Rapoport, T.A., Pohl, P. (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell.2. Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem.3. Knyazev, D.G., Winter, L., Bauer, B.W., Siligan, C., Pohl, P. (2014). Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation. J. Biol. Chem.

Signal-sequence induced conformational changes in the signal recognition particle.

Co-translational protein targeting is an essential, evolutionarily conserved pathway for delivering nascent proteins to the proper cellular membrane. In this pathway, the signal recognition particle (SRP) first recognizes the N-terminal signal sequence of nascent proteins and subsequently interacts with the SRP receptor. For this, signal sequence binding in the SRP54 M domain must be effectively communicated to the SRP54 NG domain that interacts with the receptor. Here we present the 2.9 Å crystal structure of unbound- and signal sequence bound SRP forms, both present in the asymmetric unit. The structures provide evidence for a coupled binding and folding mechanism in which signal sequence binding induces the concerted folding of the GM linker helix, the finger loop, and the C-terminal alpha helix αM6. This mechanism allows for a high degree of structural adaptability of the binding site and suggests how signal sequence binding in the M domain is coupled to repositioning of the NG domain.

Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2

Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded by both mitochondrial and nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 and the tissue-specific (predominantly lung) isoform 2 (COX4I2). COX4I2 renders lung COX about 2-fold more active compared with liver COX, which lacks COX4I2. We previously identified a highly conserved 13-bp sequence in the proximal promoter of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at a 4% oxygen concentration. Here, we have identified three transcription factors that bind this conserved ORE, namely recombination signal sequence–binding protein Jκ (RBPJ), coiled-coil-helix-coiled-coil-helix domain 2 (CHCHD2) and CXXC finger protein 5 (CXXC5). We demonstrate that RBPJ and CHCHD2 function towards activating the ORE at 4% oxygen, whereas CXXC5 functions as an inhibitor. To validate results derived from cultured cells, we show using RNA interference a similar effect of these transcription factors in the gene regulation of COX4I2 in primary pulmonary arterial smooth muscle cells. Depending on the oxygen tension, a concerted action of the three transcription factors regulates the expression of COX4I2 that, as we discuss, could augment both COX activity and its ability to cope with altered cellular energy requirements.

Fingerloop activates cargo delivery and unloading during cotranslational protein targeting

During cotranslational protein targeting by the signal recognition particle (SRP), information about signal sequence binding in the SRP's M domain must be effectively communicated to its GTPase domain to turn on its interaction with the SRP receptor (SR) and thus deliver the cargo proteins to the membrane. A universally conserved "fingerloop" lines the signal sequence-binding groove of SRP; the precise role of this fingerloop in protein targeting has remained elusive. In this study, we show that the fingerloop plays important roles in SRP function by helping to induce the SRP into a more active conformation that facilitates multiple steps in the pathway, including efficient recruitment of SR, GTPase activation in the SRP•SR complex, and most significantly, the unloading of cargo onto the target membrane. On the basis of these results and recent structural work, we propose that the fingerloop is the first structural element to detect signal sequence binding; this information is relayed to the linker connecting the SRP's M and G domains and thus activates the SRP and SR for carrying out downstream steps in the pathway.

Carboxyl-Terminal Amino Acids 1052 to 1082 of the Latency-Associated Nuclear Antigen (LANA) Interact with RBP-Jκ and Are Responsible for LANA-Mediated RTA Repression

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is closely associated with several malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV can establish lifelong latency in the host, but the mechanism is not fully understood. Previous studies have proposed a feedback model in which the viral replication and transcription activator (RTA) can induce the expression of the latency-associated nuclear antigen (LANA) during early infection. LANA, in turn, represses transcription and RTA function to establish and maintain KSHV latency. The interaction between LANA and the recombination signal sequence binding protein Jκ (RBP-Jκ, also called CSL), a major transcriptional repressor of the Notch signaling pathway, is essential for RTA repression. In the present study, we show that the LANA carboxyl-terminal amino acids 1052 to 1082 are responsible for the LANA interaction with RBP-Jκ. The secondary structure of the LANA carboxyl terminus resembles the RBP-Jκ-associated module (RAM) of Notch receptor. Furthermore, deletion of the region of LANA residues 1052 to 1082 resulted in aberrant expression of RTA, leading to elevated viral lytic replication. For the first time, we dissected a conserved RBP-Jκ binding domain in LANA and demonstrated that this domain was indispensable for LANA-mediated repression of KSHV lytic genes, thus helping the virus maintain latency and control viral reactivation.

Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles, which reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity.

Structural basis of signal-sequence recognition by the signal recognition particle

The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain-structural changes that ensure a hierarchical succession of events during protein targeting.

Mechanisms of protein translocation across membranes

Many proteins in bacteria are transported during their biosynthesis across or are integrated into the plasma membrane, a process that is similar to protein translocation across endoplasmic reticulum (ER) membrane in eukaryotes. Transport occurs through a protein‐conducting channel that is formed from a conserved heterotrimeric membrane protein complex (SecY or Sec61 complex). The channel associates with different partners in different translocation pathways. In bacteria, the SecY channel can associate with the translating ribosome (co‐translational translocation) or with the cytoplasmic ATPase SecA (post‐translational translocation).The crystal structure of an archaeal SecY complex shows the architecture of the closed channel. The structure suggests that a single SecY copy forms the translocation pore, a prediction that was confirmed for SecA‐mediated translocation by disulfide bridge crosslinking experiments. However, translocation appears to be mediated by oligomers of the SecY complex; nucleotide‐binding fold 1 (NBF1) of SecA interacts with a non‐translocating SecY copy, while other domains of SecA "push" the polypeptide chain through a neighboring SecY copy.A recently determined crystal structure shows one copy of SecA in its transitions state of ATP hydrolysis bound to one copy of SecY complex. The latter corresponds to the translocating SecY copy. Both SecA and SecY undergo major conformational changes upon interaction. The structure suggests mechanisms for how SecA moves polypeptides through the SecY channel. In addition, the data reveal that SecA binding opens the lateral gate of SecY for signal sequence intercalation, and induces plug movement. These results indicate that the channel is opened for translocation in two distinct steps, induced by SecA and signal sequence binding.

Structures of SRP54 and SRP19, the Two Proteins that Organize the Ribonucleic Core of the Signal Recognition Particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 Å resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely α-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 Å resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19•SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.

Structure of a complex of the ATPase SecA and the protein-translocation channel

Most proteins are secreted from bacteria by the interaction of the cytoplasmic SecA ATPase with a membrane channel, formed by the heterotrimeric SecY complex. Here we report the crystal structure of SecA bound to the SecY complex, with a maximum resolution of 4.5 ångström (A), obtained for components from Thermotoga maritima. One copy of SecA in an intermediate state of ATP hydrolysis is bound to one molecule of the SecY complex. Both partners undergo important conformational changes on interaction. The polypeptide-cross-linking domain of SecA makes a large conformational change that could capture the translocation substrate in a 'clamp'. Polypeptide movement through the SecY channel could be achieved by the motion of a 'two-helix finger' of SecA inside the cytoplasmic funnel of SecY, and by the coordinated tightening and widening of SecA's clamp above the SecY pore. SecA binding generates a 'window' at the lateral gate of the SecY channel and it displaces the plug domain, preparing the channel for signal sequence binding and channel opening.

Roles for Srp72p in assembly, nuclear export and function of the signal recognition particle

Co-translational protein targeting to the endoplasmic reticulum is catalysed by the signal recognition particle, a conserved ribonucleoprotein. Key activities of SRP – signal sequence binding, and inhibition of ribosomal translation elongation – require interactions of SRP with distant locations on the ribosome. A heterodimer of Srp72p and Srp68p localise to the central portion of the SRP complex, and may co-ordinate its activities. A series of mutations within Srp72p were examined individually, in combination and in the presence of mutations within SRP RNA. In this analysis mutations within Srp72p fell into two groups, identifying separate interactions/functions of the protein. Much of Srp72p is predicted to be alpha helical tetratricopeptide repeat motifs, with the C-terminus forming a separate unstructured region. Mutations towards the C-terminal end of the alpha helical region reveal a specific genetic interaction with a conserved motif in the central helix of SRP RNA. In contrast, mutations within the C-terminal region of Srp72p have genetic interactions across the RNA. Many mutant combinations impaired function rather than inhibiting assembly of SRP. However, one specific combination of Srp72p and SRP RNA mutations led to accumulation of pre-SRP in the nucleus. We conclude that that Srp72p has at least two functions that are individually redundant and that the conformation of the complex is critical for efficient completion of its biogenesis.

Role of Sox2 in the development of the mouse neocortex

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle. Overexpression of constitutively active Sox2 in neural progenitors resulted in upregulation of Notch1, recombination signal-sequence binding protein-J (RBP-J) and hairy enhancer of split 5 (Hes5) transcripts and the Sox2 high mobility group (HMG) domain seemed sufficient to confer these effects. While Sox2 overexpression permitted the differentiation of progenitors into astroglia, it inhibited neurogenesis, unless the Notch pathway was blocked. Moreover, neuronal precursors engaged a serine protease(s) to eliminate the overexpressed Sox2 protein and relieve the repression of neurogenesis. Glial precursors and differentiated astrocytes, on the other hand, maintained Sox2 expression until they reached a quiescent state. Sox2 expression was re-activated by signals that triggered astrocytic proliferation (i.e., injury, mitogenic and gliogenic factors). Taken together, Sox2 appears to act upstream of the Notch signaling pathway to maintain the cell proliferative potential and to ensure the generation of sufficient cell numbers and phenotypes in the developing neocortex.

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.

Investigating the SecY plug movement at the SecYEG translocation channel

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

Modeling the Effects of prl Mutations on the Escherichia coli SecY Complex

The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the prl suppressor alleles of secE and secY, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in secE and secY, as well as the phenotypic effects of combinations of prl mutations. These new alleles, as well as previously characterized prl mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the prlA and prlG alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex.

Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jkappa (RBP-Jkappa), as a RBP-Jkappa-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jkappa. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

Kaposi's Sarcoma-Associated Herpesvirus Reactivation Is Regulated by Interaction of Latency-Associated Nuclear Antigen with Recombination Signal Sequence-Binding Protein Jκ, the Major Downstream Effector of the Notch Signaling Pathway

Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA controls KSHV latent infection through repression of RTA, an activator of many lytic promoters. RTA activates the expression of several lytic viral genes by interacting with recombination signal sequence-binding protein Jkappa (RBP-Jkappa), a transcriptional repressor and the target of the Notch signaling pathway. The recognition that a number of KSHV lytic gene promoters, including RTA, contain RBP-Jkappa binding sites raised the possibility that RBP-Jkappa-mediated repression may be central to the establishment of latency. Here, we tested this hypothesis by examining the regulation of RTA by LANA through binding to RBP-Jkappa. This study demonstrates that LANA physically associates with RBP-Jkappa in vitro and in KSHV-infected cells, with the complex formed capable of binding to RBP-Jkappa cognate sequences. RBP-Jkappa binding sites within the RTA promoter have been found to be critical for LANA-mediated repression. Our study describes a novel mechanism through which LANA maintains KSHV latency by targeting a major downstream effector of the Notch signaling pathway.

Heterodimeric GTPase core of the SRP targeting complex.

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

Mapping the Signal Sequence-binding Site on SRP Reveals a Significant Role for the NG Domain

We present evidence that the signal recognition particle (SRP) recognizes signal sequences via the NG domain on the SRP54 protein subunit. Using a recently developed cross-linking method (Fancy, D. A., and Kodadek, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6020-6024; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1317), we find that signal peptides cross-link to the Escherichia coli SRP protein Ffh (the homologue of the mammalian SRP54 subunit) via the NG domain. Within the NG domain, the cross-linking site maps to the ras-like C-terminal subdomain termed the G domain. This result stands in contrast to previous studies, which concluded based on nascent chain cross-linking that the signal sequence bound to the adjacent M domain. As independent evidence of a direct binding interaction between the NG domain and the signal sequence, we find that the NG domain of Ffh binds signal peptides as an isolated entity. Our results suggest that the NG domain forms a substantial part of the binding site for the signal sequence.