Specific Targeting Signals Research Articles

Palindrome sequence mediated target recycling integrating with self-priming assisted signal reaction for sensitive miRNA detection

The development of a sensitive and isothermal technique with a greatly enhanced miRNA detection signal is still technically problematic due to the low abundance of miRNA and high sequence similarities with homologous miRNAs. Herein, we propose a novel fluorescence approach for sensitive and reliable miRNA detection by integrating the palindrome sequence mediated target recycling with self-priming assisted signal reaction. In this method, a dual toehold DNA nano-probe (HT) with two functional arms is designed to mediate specific target recognition and signal amplification. In the presence of target miRNA, it binds to the recognition module of HT probe, releasing the “2” sequence to initiate strand displacement amplification (SDA) and a self-priming-induced signal reaction. Based on the elegant design, the proposed method exhibits a wide linear response range exceeding five orders of magnitude and a low limit of detection of 0.96 fM according to the 3δ rule. The non-specific signal is below 5 % for non-target miRNA detection. Taking the merits of excellent sensitivity, desirable specificity, and superior anti-interference ability, the proposed approach shows a promising prospect for detecting miRNAs in complicated biological environments and early diagnosis of diseases.

In vitro import of mitochondrial precursor proteins into yeast mitochondria.

Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.

Protein transport along the presequence pathway.

Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes- TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.

Protein translocation in mitochondria: Sorting out the Toms, Tims, Pams, Sams and Mia.

Mitochondria contain 902 (yeast) to 1.136 (mouse, humans) verified proteins. Except for a very small number of mitochondrially encoded core components of the respiratory chain, mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol. Different import pathways direct proteins to their respective mitochondrial subcompartment (outer membrane, intermembrane space (IMS), inner membrane and matrix). Specific targeting signals in their sequence direct proteins to their target destination and allow the proteins to embark on their respective import pathway. The main import pathways are shown here on the poster and are introduced in the following, using the mitochondrial import system of the baker's yeast Saccharomyces cerevisiae as example. However, the mitochondrial import system of mammalian cells is highly similar and deviates only in minor aspects. Even the mitochondrial import machineries of less closely related eukaryotes, such as plants and trypanosomes, are very similar and adhere to the same general principles.

Targeting of Proteins for Translocation at the Endoplasmic Reticulum.

The endoplasmic reticulum represents the gateway to the secretory pathway. Here, proteins destined for secretion, as well as soluble and membrane proteins that reside in the endomembrane system and plasma membrane, are triaged from proteins that will remain in the cytosol or be targeted to other cellular organelles. This process requires the faithful recognition of specific targeting signals and subsequent delivery mechanisms to then target them to the translocases present at the ER membrane, which can either translocate them into the ER lumen or insert them into the lipid bilayer. This review focuses on the current understanding of the first step in this process representing the targeting phase. Targeting is typically mediated by cleavable N-terminal hydrophobic signal sequences or internal membrane anchor sequences; these can either be captured co-translationally at the ribosome or recognised post-translationally and then delivered to the ER translocases. Location and features of the targeting sequence dictate which of several overlapping targeting pathway substrates will be used. Mutations in the targeting machinery or targeting signals can be linked to diseases.

Coordinated Translocation of Presequence-Containing Precursor Proteins Across Two Mitochondrial Membranes: Knowns and Unknowns of How TOM and TIM23 Complexes Cooperate With Each Other.

The vast majority of mitochondrial proteins are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals. Mitochondrial targeting signals are very diverse, however, about 70% of mitochondrial proteins carry cleavable, N-terminal extensions called presequences. These amphipathic helices with one positively charged and one hydrophobic surface target proteins to the mitochondrial matrix with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. Translocation of proteins across the two mitochondrial membranes does not take place independently of each other. Rather, in the intermembrane space, where the two complexes meet, components of the TOM and TIM23 complexes form an intricate network of protein–protein interactions that mediates initially transfer of presequences and then of the entire precursor proteins from the outer to the inner mitochondrial membrane. In this Mini Review, we summarize our current understanding of how the TOM and TIM23 complexes cooperate with each other and highlight some of the future challenges and unresolved questions in the field.

(E)-Guggulsterone Inhibits Dengue Virus Replication by Upregulating Antiviral Interferon Responses through the Induction of Heme Oxygenase-1 Expression.

Dengue virus (DENV) infection, which causes dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is a severe global health problem in tropical and subtropical areas. There is no effective vaccine or drug against DENV infection. Thus, the development of anti-DENV agents is imperative. This study aimed to assess the anti-DENV activity of (E)-guggulsterone using a DENV infectious system. A specific inhibitor targeting signal molecules was used to evaluate the molecular mechanisms of action. Western blotting and qRT-PCR were used to determine DENV protein expression and RNA replication, respectively. Finally, an ICR suckling mouse model was used to examine the anti-DENV activity of (E)-guggulsterone in vivo. A dose-dependent inhibitory effect of (E)-guggulsterone on DENV protein synthesis and RNA replication without cytotoxicity was observed. The mechanistic studied revealed that (E)-guggulsterone stimulates Nrf2-mediated heme oxygenase-1 (HO-1) expression, which increases the antiviral interferon responses and downstream antiviral gene expression by blocking DENV NS2B/3B protease activity. Moreover, (E)-guggulsterone protected ICR suckling mice from life-threatening DENV infection. These results suggest that (E)-guggulsterone can be a potential supplement for controlling DENV replication.

Versatile allosteric properties in Pex5-like tetratricopeptide repeat proteins to induce diverse downstream function.

Proteins composed of tetratricopeptide repeat (TPR) arrays belong to the α-solenoid tandem-repeat family that have unique properties in terms of their overall conformational flexibility and ability to bind to multiple protein ligands. The peroxisomal matrix protein import receptor Pex5 comprises two TPR triplets that recognize protein cargos with a specific C-terminal Peroxisomal Targeting Signal (PTS) 1 motif. Import of PTS1-containing protein cargos into peroxisomes through a transient pore is mainly driven by allosteric binding, coupling and release mechanisms, without a need for external energy. A very similar TPR architecture is found in the functionally unrelated TRIP8b, a regulator of the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. TRIP8b binds to the HCN ion channel via a C-terminal sequence motif that is nearly identical to the PTS1 motif of Pex5 receptor cargos. Pex5, Pex5-related Pex9, and TRIP8b also share a less conserved N-terminal domain. This domain provides a second protein cargo-binding site and plays a distinct role in allosteric coupling of initial cargo loading by PTS1 motif-mediated interactions and different downstream functional readouts. The data reviewed here highlight the overarching role of molecular allostery in driving the diverse functions of TPR array proteins, which could form a model for other α-solenoid tandem-repeat proteins involved in translocation processes across membranes.

Development of an Antisense Oligonucleotide-Mediated Exon Skipping Therapeutic Strategy for Mucolipidosis II: Validation at RNA Level.

Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.

Multiple Localization by Functional Translational Readthrough.

In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as astarting point, proteins are imported into organelles by specific targeting signals. Many proteins, however, act in more than one cellular compartment. In this chapter, we discuss mechanisms by which proteins can be targeted to multiple organelles with a focus on a novel gene regulatory mechanism, functional translational readthrough, that permits multiple targeting of proteins to the peroxisome and other organelles. In mammals, lactate and malate dehydrogenase are the best-characterized enzymes whose targeting is controlled by functional translational readthrough.

In Silico Tools for the Prediction of Protein Import into Secondary Plastids.

The in silico identification of proteins targeting to secondary plastids is a difficult task. Such plastids are complex in structure and can be surrounded by up to four membranes, which have to be crossed during import. Nucleus-encoded plastidial preproteins in organisms with secondary plastids contain specific N-terminal targeting signals, the so-called bipartite targeting signal (BTS) sequences consisting of a classical signal peptide followed by a transit peptide-like sequence, mediating this intricate process. As these signal sequences differ significantly from transit peptides of plastid preproteins in plants and other organisms with primary plastids, existing in silico tools for primary plastid targeting prediction are not directly suitable to detect nucleus-encoded proteins destined for the import into secondary plastids. In this chapter I describe the current state-of-the-art methods to reliably predict proteins that might be imported into secondary plastids of red- and green-algal origin using either the "classical" approach, which involves a combination of bits of information produced by existing in silico tools, or, if available, via consulting specifically developed algorithms.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Abstract P4-02-14: PD-1-IRDye800CW as a novel probe for fluorescence molecular imaging and image guided intraoperative surgery in breast tumor mouse model

Abstract P4-02-14: PD-1-IRDye800CW as a novel probe for fluorescence molecular imaging and image guided intraoperative surgery in breast tumor mouse model

Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact Mammalian Cell System.

Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including β-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.

Microcavity-Integrated Carbon Nanotube Photodetectors.

Carbon nanotubes (CNTs) are considered to be highly promising nanomaterials for multiwavelength, room-temperature infrared detection applications. Here, we demonstrate a single-tube diode photodetector monolithically integrated with a Fabry-Pérot microcavity. A ∼6-fold enhanced optical absorption can be achieved, because of the confined effect of the designed optical mode. Furthermore, taking advantage of Van-Hove-singularity band structures in CNTs, we open the possibility of developing chirality-specific (n,m) CNT-film-based signal detectors. Utilizing a concept of the "resonance and off-resonance" cavity, we achieved cavity-integrated chirality-sorted CNT-film detectors working at zero bias and resonance-allowed mode, for specific target signal detection. The detectors exhibited a higher suppression ratio until a power density of 0.07 W cm(-2) and photocurrent of 5 pA, and the spectral full width at half-maximum is ∼33 nm at a signal wavelength of 1200 nm. Further, with multiple array detectors aiming at different target signals integrated on a chip, a multiwavelength signal detector system can be expected to have applications in the fields of monitoring, biosensing, color imaging, signal capture, and on-chip or space information transfers. The approach can also bring other nanomaterials into on-chip or information optoelectronics, regardless of the available doping polarity.

Multichannel Signal Separation Combining Directional Clustering and Nonnegative Matrix Factorization with Spectrogram Restoration

In this paper, to address problems in multichannel music signal separation, we propose a new hybrid method that combines directional clustering and advanced nonnegative matrix factorization (NMF). The aims of multichannel music signal separation technology is to extract a specific target signal from observed multichannel signals that contain multiple instrumental sounds. In previous studies, various methods using NMF have been proposed, but many problems remain including poor separation accuracy and lack of robustness. To solve these problems, we propose a new supervised NMF (SNMF) with spectrogram restoration and a hybrid method that concatenates the proposed SNMF after directional clustering. Via the extrapolation of supervised spectral bases, the proposed SNMF attempts both target signal separation and reconstruction of the lost target components, which are generated by preceding directional clustering. In addition, we experimentally reveal the trade-off between separation and extrapolation abilities and propose a new scheme for adaptive divergence, where the optimal divergence can be automatically changed in each time frame according to the local spatial conditions. The results of an evaluation experiment show that our proposed hybrid method outperforms the conventional music signal separation methods.

Reconsidering ideas regarding the evolution of peroxisomes: the case for a mitochondrial connection.

oxygen radical formation during fatty acid metabolism leads to the selective force favouring peroxisome formation. Dr. Gabaldon believes that the cellular site is the er, while I proposed the (pre)mitochondrion. this brings me to an important part of his article: his (implicit) arguments against my scenario. the most fundamental peroxisomal pathway is beta oxidation [its 4 steps catalysed sequentially by: an oxidase (step 1), a hydratase (step 2), a dehydrogenase (step 3) and a thiolase (step 4); performed by only three enzymes, with step 2 and 3 both being catalysed by a single protein, namely, Pox2p in yeast]. the three enzymes are all needed for complete oxidation, so the use of an “alpha-proteobacterial” Pox2p would strongly suggest the presence of the (pre)mitochondrion when peroxisomes were evolving. Surprisingly, the opposite conclusion is drawn by Dr. Gabaldon: “Hence, in contrast to earlier suggestions [25], this model does not necessarily imply that mitochondrial endosymbiosis pre-dated the origin of peroxisomes.” Of note, the “[25]” refers to my article— and is the only reference to my article. the presence of a “host” Pox1p for the first breakdown step is irrelevant in this regard, as it does not allow us to see whether the endosymbiont was already present or not. also the next argument, namely, that the direct er model is simpler because “...coupling the re-targeting of mitochondrial enzymes to the formation of peroxisomes implies that these enzymes were targeted first to the er...”, is unconvincing. First of all, such re-targeting has been documented many times, showing it to be no big hurdle. Secondly, new endomembrane formations, such as the nucleus, the er and peroxisomes, can be seen as “reactions” to the problems generated by endosymbiont entry; consequently, the presence of a full blown er with specific targeting signals when peroxisomes originated is not that likely. Stressing adaptations to the endosymbiont as the basis of the eukaryotic the article by toni Gabaldon entitled “a metabolic scenario for the evolutionary origin of peroxisomes from the endomembranous system” that recently appeared in Cellular and Molecular Life Sciences [1] left me somewhat confused. In this article, the author proposes a model for the evolutionary origins of peroxisomes that can be described as follows: (1) peroxisomes are derived from the endoplasmic reticulum (er); (2) the evolutionary forces driving the separation of peroxisomes from the er stem from fatty acid metabolism in which certain reactions produce highly reactive toxic species, providing a strong selective force in favour of sequestering these reactions. Dr. Gabaldon states that such a hypothesis on the endomembranous origin of peroxisomes “has never been formally articulated in the context of a plausible evolutionary scenario describing the possible selective forces involved”. However, in 2010 I published an article which, in light of Dr. Gabaldon’s recent publication, has a surprising title: “Oxygen radicals shaping evolution: why fatty acid catabolism leads to peroxisomes while neurons do without it: FaDH2/naDH flux ratios determining mitochondrial radical formation were crucial for the eukaryotic invention of peroxisomes and catabolic tissue differentiation” [2]. as can be garnered from the title, the description of Dr. Gabaldon’s model given above, matches the model in my article (“eukaryotic invention” refers to the fact that peroxisomes coevolved with/are derived from the er). are there differences? Yes, apart from shared characteristics, there is one fundamental difference between the two incarnations of the model: the cellular site at which

Congenital Sucrase‐Isomaltase Deficiency

Congenital Sucrase‐Isomaltase Deficiency

Differentiation and Visualization of Diverse Cellular Phenotypic Responses in Primary High-Content Screening

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating “scaffold hopping.”

Investigating Synaptophysin Targeting to the Photoreceptor Synapse

Synaptophysin (SYP) is the most abundant protein found in synaptic vesicles. It functions to regulate the endocytosis phase of the synaptic vesicle cycle. However, it is not known how newly synthesized SYP is targeted from the neuronal soma to the synapse so that it can carry out this function. The photoreceptor is an excellent neuron in which to tackle this problem, due in part to its large size and clear compartmentalization. The objective of this study is to test the hypothesis that SYP contains a sequence specific targeting signal that directs the targeting of this protein to the synaptic terminal in photoreceptors. Our approach was to generate constructs consisting of a membrane‐associated YFP as a reporter fused to various regions of SYP, and express those constructs in the rod photoreceptors of transgenic Xenopus laevis. The subcellular localization of these proteins in tadpole photoreceptors was assessed by confocal microscopy. The YFP reporter was found throughout the cell but strongly accumulated in the outer segment, as we have previously described for membrane proteins lacking targeting information in this cell type. We found that only the cytoplasmic C‐terminus of SYP was capable of restricting the localization of the reporter to the inner segment plasma membrane and synaptic terminal. Future studies are aimed at determining which of SYP's binding partners regulates its trafficking in photoreceptors.