Precise subcellular targeting approaches for organelle-related disorders.

Pharmacological research has expanded to the nanoscale level with advanced imaging technologies, enabling the analysis of drug distribution at the cellular organelle level. These advances in research techniques have contributed to the targeting of cellular organelles to address the fundamental causes of diseases. Beyond navigating the hurdles of reaching lesion tissues upon administration and identifying target cells within these tissues, controlling drug accumulation at the organelle level is the most refined method of disease management. This approach opens new avenues for the development of more potent therapeutic strategies by delving into the intricate roles and interplay of cellular organelles. Thus, organelle-targeted approaches help overcome the limitations of conventional therapies by precisely regulating functionally compartmentalized spaces based on their environment. This review discusses the basic concepts of organelle targeting research and proposes strategies to target diseases arising from organelle dysfunction. We also address the current challenges faced by organelle targeting and explore future research directions.

Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using "2A" Self-Cleaving Peptides.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or "gene stacking") have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

Unlocking the Mitochondria for Nanomedicine-based Treatments: Overcoming Biological Barriers, Improving Designs, and Selecting Verification Techniques.

Enhanced targeting approaches will support the treatment of diseases associated with dysfunctional mitochondria, which play critical roles in energy generation and cell survival. Obstacles to mitochondria-specific targeting include the presence of distinct biological barriers and the need to pass through (or avoid) various cell internalization mechanisms. A range of studies have reported the design of mitochondrially-targeted nanomedicines that navigate the complex routes required to influence mitochondrial function; nonetheless, a significant journey lies ahead before mitochondrially-targeted nanomedicines become suitable for clinical use. Moving swiftly forward will require safety studies, in vivo assays confirming effectiveness, and methodologies to validate mitochondria-targeted nanomedicines' subcellular location/activity. From a nanomedicine standpoint, we describe the biological routes involved (from administration to arrival within the mitochondria), the features influencing rational design, and the techniques used to identify/validate successful targeting. Overall, rationally-designed mitochondria-targeted-based nanomedicines hold great promise for precise subcellular therapeutic delivery.

Expressing Optogenetic Actuators Fused to N-terminal Mucin Motifs Delivers Targets to Specific Subcellular Compartments in Polarized Cells.

Optogenetics is a powerful approach in neuroscience research. However, other tissues of the body may benefit from controlled ion currents and neuroscience may benefit from more precise optogenetic expression. The present work constructs three subcellularly-targeted optogenetic actuators based on the channelrhodopsin ChR2-XXL, utilizing 5, 10, or 15 tandem repeats (TR) from mucin as N-terminal targeting motifs and evaluates expression in several polarized and non-polarized cell types. The modified channelrhodopsin maintains its electrophysiological properties, which can be used to produce continuous membrane depolarization, despite the expected size of the repeats. This work then shows that these actuators are subcellularly localized in polarized cells. In polarized epithelial cells, all three actuators localize to just the lateral membrane. The TR-tagged constructs also express subcellularly in cortical neurons, where TR5-ChR2XXL and TR10-ChR2XXL mainly target the somatodendrites. Moreover, the transfection efficiencies are shown to be dependent on cell type and tandem repeat length. Overall, this work verifies that the targeting motifs from epithelial cells can be used to localize optogenetic actuators in both epithelia and neurons, opening epithelia processes to optogenetic manipulation and providing new possibilities to target optogenetic tools.

Cell-permeable peptide-based delivery vehicles useful for subcellular targeting and beyond.

Personal medicine aims to provide tailor-made diagnostics and treatments and has been emerged as a promising but challenging strategy during the last years. This includes the active delivery and localization of a therapeutic compound to a targeted site of action within a cell. An example being targeting the interference of a distinct protein-protein interaction (PPI) within the cell nucleus, mitochondria or other subcellular location. Therefore, not only the cell membrane has to be overcome but also the final intracellular destination has to be reached. One approach which fulfills both requirements is to use short peptide sequences that are able to translocate into cells as targeting and delivery vehicles. In fact, recent progress in this field demonstrates how these tools can modulate the pharmacological parameters of a drug without compromising its biological activity. Beside classical targets that are addressed by various small molecule drugs such as receptors, enzymes, or ion channels, PPIs have received increasing attention as potential therapeutic targets. Within this review, we will provide a recent update on cell-permeable peptides targeting subcellular destinations. We include chimeric peptide probes that combine cell-penetrating peptides (CPPs) and a targeting sequence, as well peptides having intrinsic cell-permeability and which are often used to target PPIs.

Novel organoboron complexes with robust core: Synthesis, functionalization, and subcellular targeting.

The construction of novel organoboron complexes with facile synthesis and unique advantages for biological imaging remains a challenge and thus has garnered considerable attention. Herein, we developed a new molecular platform, boron indolin-3-one-pyrrol (BOIN3OPY) via a two-step sequential reaction. The molecular core is robust enough to allow for post-functionalization to produce versatile dyes. When compared to the standard BODIPY, these dyes feature an N,O-bidentate seven-membered ring center, significantly redshifted absorption, and a larger Stokes shift. This study establishes a new molecular platform that provides more flexibility for the functional regulation of dyes.

Peroxidase proximity selection to identify aptamers targeting a subcellular location.

The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.

Activation of pyroptosis by specific organelle-targeting photodynamic therapy to amplify immunogenic cell death for anti-tumor immunotherapy.

Pyroptosis, a unique lytic programmed cell death, inspired tempting implications as potent anti-tumor strategy in pertinent to its potentials in stimulating anti-tumor immunity for eradication of primary tumors and metastasis. Nonetheless, rare therapeutics have been reported to successfully stimulate pyroptosis. In view of the intimate participation of reactive oxygen species (ROS) in stimulating pyroptosis, we attempted to devise a spectrum of well-defined subcellular organelle (including mitochondria, lysosomes and endoplasmic reticulum)-targeting photosensitizers with the aim of precisely localizing ROS (produced from photosensitizers) at the subcellular compartments and explore their potentials in urging pyroptosis and immunogenic cell death (ICD). The subsequent investigations revealed varied degrees of pyroptosis upon photodynamic therapy (PDT) towards cancerous cells, as supported by not only observation of the distinctive morphological and mechanistic characteristics of pyroptosis, but for the first-time explicit validation from comprehensive RNA-Seq analysis. Furthermore, in vivo anti-tumor PDT could exert eradication of the primary tumors, more importantly suppressed the distant tumor and metastatic tumor growth through an abscopal effect, approving the acquirement of specific anti-tumor immunity as a consequence of pyroptosis. Hence, pyroptosis was concluded unprecedently by our proposed organelles-targeting PDT strategy and explicitly delineated with molecular insights into its occurrence and the consequent ICD.

Aminoacyl-tRNA Synthetase Evolution within the Dynamic Tripartite Translation System of Plant Cells.

Eukaryotes maintain separate protein translation systems for nuclear and organellar genes, including distinct sets of tRNAs and aminoacyl-tRNA synthetases (aaRSs). In animals, mitochondrial-targeted aaRSs are expressed at lower levels and are less conserved in sequence than cytosolic aaRSs involved in translation of nuclear mRNAs, likely reflecting lower translational demands in mitochondria. In plants, translation is further complicated by the presence of plastids, which share most aaRSs with mitochondria. In addition, plant mitochondrial tRNA pools have a dynamic history of gene loss and functional replacement by tRNAs from other compartments. To investigate the consequences of these distinctive features of translation in plants, we analyzed sequence evolution in angiosperm aaRSs. In contrast to previously studied eukaryotic systems, we found that plant organellar and cytosolic aaRSs exhibit only a small difference in expression levels, and organellar aaRSs are slightly more conserved than cytosolic aaRSs. We hypothesize that these patterns result from high translational demands associated with photosynthesis in mature chloroplasts. We also investigated aaRS evolution in Sileneae, an angiosperm lineage with extensive mitochondrial tRNA replacement and aaRS retargeting. We predicted positive selection for changes in aaRS sequence resulting from these recent changes in subcellular localization and tRNA substrates but found little evidence for accelerated sequence divergence. Overall, the complex tripartite translation system in plant cells appears to have imposed more constraints on the long-term evolutionary rates of organellar aaRSs compared with other eukaryotic lineages, and plant aaRS protein sequences appear largely robust to more recent perturbations in subcellular localization and tRNA interactions.

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

The Golgi Apparatus as an Anticancer Therapeutic Target.

Although the discovery of the Golgi apparatus (GA) was made over 125 years ago, only a very limited number of therapeutic approaches have been developed to target this complex organelle. The GA serves as a modification and transport center for proteins and lipids and also has more recently emerged as an important store for some ions. The dysregulation of GA functions is implicated in many cellular processes associated with cancer and some GA proteins are indeed described as cancer biomarkers. This dysregulation can affect protein modification, localization, and secretion, but also cellular metabolism, redox status, extracellular pH, and the extracellular matrix structure. Consequently, it can directly or indirectly affect cancer progression. For these reasons, the GA is an appealing anticancer pharmacological target. Despite this, no anticancer drug specifically targeting the GA has reached the clinic and few have entered the clinical trial stage. Advances in nanodelivery approaches may help change this scenario by specifically targeting tumor cells and/or the GA through passive, active, or physical strategies. This article aims to examine the currently available anticancer GA-targeted drugs and the nanodelivery strategies explored for their administration. The potential benefits and challenges of modulating and specifically targeting the GA function in the context of cancer therapy are discussed.

The Evolutionary History of Peptidases Involved in the Processing of Organelle-Targeting Peptides.

Most of the proteins present in mitochondria and chloroplasts, the organelles acquired via endosymbiotic events, are encoded in the nucleus and translated into the cytosol. Most of such nuclear-encoded proteins are specifically recognized via an N-terminal-encoded targeting peptide (TP) and imported into the organelles via a translocon machinery. Once imported, the TP is degraded by a succession of cleavage steps ensured by dedicated peptidases. Here, we retrace the evolution of the families of the mitochondrial processing peptidase (MPP), stromal processing peptidase (SPP), presequence protease (PreP), and organellar oligo-peptidase (OOP) that play a central role in TP processing and degradation across the tree of life. Their bacterial distributions are widespread but patchy, revealing unsurprisingly complex history of lateral transfers among bacteria. We provide evidence for the eukaryotic acquisition of MPP, OOP, and PreP by lateral gene transfers from bacteria at the time of the mitochondrial endosymbiosis. We show that the acquisition of SPP and of a second copy of OOP and PreP at the time of the chloroplast endosymbiosis was followed by a differential loss of one PreP paralog in photosynthetic eukaryotes. We identified some contrasting sequence conservations between bacterial and eukaryotic homologs that could reflect differences in the functional context of their peptidase activity. The close vicinity of the eukaryotic peptidases MPP and OOP to those of several bacterial pathogens, showing antimicrobial resistance, supports a scenario where such bacteria were instrumental in the establishment of the proteolytic pathway for TP degradation in organelles. The evidence for their role in the acquisition of PreP is weaker, and none is observed for SPP, although it cannot be excluded by the present study.

Subcellular delivery of lipid nanoparticles to endoplasmic reticulum and mitochondria.

Primarily responsible for the biogenesis and metabolism of biomolecules, endoplasmic reticulum (ER) and mitochondria are gradually becoming the targets of therapeutic modulation, whose physiological activities and pathological manifestations determine the functional capacity and even the survival of cells. Drug delivery systems with specific physicochemical properties (passive targeting), or modified by small molecular compounds, polypeptides, and biomembranes demonstrating tropism for ER and mitochondria (active targeting) are able to reduce the nonselective accumulation of drugs, enhancing efficacy while reducing side effects. Lipid nanoparticles feature high biocompatibility, diverse cargo loading, and flexible structure modification, which are frequently used for subcellular organelle-targeted delivery of therapeutics. However, there is still a lack of systematic understanding of lipid nanoparticle-based ER and mitochondria targeting. Herein, we review the pathological significance of drug selectively delivered to the ER and mitochondria. We also summarize the molecular basis and application prospects of lipid nanoparticle-based ER and mitochondria targeting strategies, which may provide guidance for the prevention and treatment of associated diseases and disorders. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Biology-Inspired Nanomaterials > Lipid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Photoactive "Bionic Virus" Robustly Elicits the Synergy Anticancer Activity of Immunophotodynamic Therapy.

Coronavirus represents an inspiring model for designing drug delivery systems due to its unique infection machinery mechanism. Herein, we have developed a biomimetic viruslike nanocomplex, termed SDN, for improving cancer theranostics. SDN has a unique core-shell structure consisting of photosensitizer chlorin e6 (Ce6)-loaded nanostructured lipid carrier (CeNLC) (virus core)@poly(allylamine hydrochloride)-functionalized MnO2 nanoparticles (virus spike), generating a virus-mimicking nanocomplex. SDN not only prompted cellular uptake through rough-surface-mediated endocytosis but also achieved mitochondrial accumulation by the interaction of cationic spikes and the anionic mitochondrial surface, leading to mitochondria-specific photodynamic therapy. Meanwhile, SDN could even mediate oxygen generation to relieve tumor hypoxia and, consequently, improve macrophage-associated anticancer immune response. Importantly, SDN served as a robust magnetic resonance imaging (MRI) contrast agent due to the fast release of Mn2+ in the presence of intracellular redox components. We identified that SDN selectively accumulated in tumors and released Mn2+ to generate a 5.71-fold higher T1-MRI signal, allowing for effectively detecting suspected tumors. Particularly, SDN induced synergistic immunophotodynamic effects to eliminate malignant tumors with minimal adverse effects. Therefore, we present a novel biomimetic strategy for improving targeted theranostics, which has a wide range of potential biomedical applications.

The difference in the intracellular Arg/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2.

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme-inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators.

cAMP controls a trafficking mechanism that maintains the neuron specificity and subcellular placement of electrical synapses.

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.

Engineering of Plants for Efficient Production of Therapeutics.

Plants are becoming useful platforms for recombinant protein production at present time. With the advancement of efficient molecular tools of genomics, proteomics, plants are now being used as a biofactory for production of different life saving therapeutics. Plant-based biofactory is an established production system with the benefits of cost-effectiveness, high scalability, rapid production, enabling post-translational modification, and being devoid of harmful pathogens contamination. This review introduces the main challenges faced by plant expression system: post-translational modifications, protein stability, biosafety concern and regulation. It also summarizes essential factors to be considered in engineering plants, including plant expression system, promoter, post-translational modification, codon optimization, and fusion tags, protein stabilization and purification, subcellular targeting, and making vaccines in an edible way. This review will be beneficial and informative to scholars and readers in the field of plant biotechnology.

Tailorable Membrane-Penetrating Nanoplatform for Highly Efficient Organelle-Specific Localization.

Given the breadth of currently arising opportunities and concerns associated with nanoparticles for biomedical imaging, various types of nanoparticles have been widely exploited, especially for cellular/subcellular level probing. However, most currently reported nanoparticles either have inefficient delivery into cells or lack specificity for intracellular destinations. The absence of well-defined nanoplatforms remains a critical challenge hindering practical nano-based bio-imaging. Herein, the authors elaborate on a tailorable membrane-penetrating nanoplatform as a carrier with encapsulated actives and decorated surfaces to tackle the above-mentioned issues. The tunable contents in such a versatile nanoplatform offer huge flexibility to reach the expected properties and functions. Aggregation-induced emission luminogen (AIEgen) is applied to achieve sought-after photophysical properties, specific targeting moieties are installed to give high affinity towards different desired organelles, and critical grafting of cell-penetrating cyclic disulfides (CPCDs) to promote cellular uptake efficiency without sacrificing the specificity. Hereafter, to validate its practicability, the tailored nano products are successfully applied to track the dynamic correlation between mitochondria and lysosomes during autophagy. The authors believe that the strategy and described materials can facilitate the development of functional nanomaterials for various life science applications.

Mitochondria and Endoplastic Reticulum Targeting Strategy for Enhanced Phototherapy.

To ensure improved efficacy and minimized toxicity of therapeutic molecules, it is generally accepted that specifically delivering them to the subcellular site of their action will be attractive. Phototherapy has received considerable attention because of its noninvasiveness, high temporal-spatial resolution, and minimal drug resistance. As important functional organelles in cells, mitochondria and endoplasmic reticulum (ER) participate in fundamental cellular processes, which make them much more sensitive to reactive oxygen species (ROS) and hyperthermia. Thus, mitochondria- or ER-targeted phototherapy will be rational strategies for synergetic cancer therapy. In this review, we focus on the latest advances in molecules and nanomaterials currently used for mitochondria- and ER-targeted phototherapy.

Mesoporous Silica Nanoparticles for Targeting Subcellular Organelles.

Current chemotherapy treatments lack great selectivity towards tumoral cells, which leads to nonspecific drug distribution and subsequent side effects. In this regard, the use of nanoparticles able to encapsulate and release therapeutic agents has attracted growing attention. In this sense, mesoporous silica nanoparticles (MSNs) have been widely employed as drug carriers owing to their exquisite physico-chemical properties. Because MSNs present a surface full of silanol groups, they can be easily functionalized to endow the nanoparticles with many different functionalities, including the introduction of moieties with affinity for the cell membrane or relevant compartments within the cell, thus increasing the efficacy of the treatments. This review manuscript will provide the state-of-the-art on MSNs functionalized for targeting subcellular compartments, focusing on the cytoplasm, the mitochondria, and the nucleus.