Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe-Functionalized Outer Surfaces.

Nanochannel technology has emerged as a powerful tool for label-free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner walls of a nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel arrays with outer-surface probes. The detection principle relies on the specific binding between the maleimide groups in outer-surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation-related diseases.

RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells.

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

Platelet membrane camouflaged AIEgen-mediated photodynamic therapy improves the effectiveness of anti-PD-L1 immunotherapy in large-burden tumors.

Although immunotherapy has achieved recent clinical success in antitumor therapy, it is less effective for solid tumors with large burdens. To overcome this challenge, herein, we report a new strategy based on platelet membrane-camouflaged aggregation-induced emission (AIE) luminogen (Plt-M@P) combined with the anti-programmed death ligand 1 (anti-PD-L1) for tumoral photodynamic-immunotherapy. Plt-M@P is prepared by using poly lactic-co-glycolic acid (PLGA)/PF3-PPh3 complex as a nanocore, and then by co-extrusion with platelet membranes. PF3-PPh3 is an AIE-active conjugated polyelectrolyte with photosensitizing capability for photodynamic therapy (PDT). Plt-M@P exhibits superior tumor targeting capacity in vivo. When applied in small tumor-bearing (~40 mm3) mice, Plt-M@P-mediated PDT significantly inhibits tumor growth. In tumor models with large burdens (~200 mm3), using Plt-M@P-mediated PDT or anti-PD-L1 alone is less effective, but the combination of both is effective in inhibiting tumor growth. Importantly, this combination therapy has good biocompatibility, as demonstrated by the absence of damage to the major organs, especially the reproductive system. In conclusion, we show that Plt-M@P-mediated PDT can improve anti-PD-L1 immunotherapy by enhancing antitumor effects, providing a promising strategy for the treatment of tumors with large burdens.

CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Novel Formulation of Undecylenic Acid induces Tumor Cell Apoptosis.

Undecylenic acid, a monounsaturated fatty acid, is currently in clinical use as a topical antifungal agent, however the potential for therapeutic application in other disease settings has not been investigated. In this study, we describe a novel platform for the solubilization of fatty acids using amino acids and utilize this approach to define a tumoricidal activity and underlying mechanism for undecylenic acid. We examined a novel formulation of undecylenic acid compounded with L-Arginine, called GS-1, that induced concentration-dependent tumor cell death, with undecylenic acid being the cytotoxic component. Further investigation revealed that GS-1-mediated cell death was caspase-dependent with a reduction in mitochondrial membrane potential, suggesting a pro-apoptotic mechanism of action. Additionally, GS-1 was found to localize intracellularly to lipid droplets. In contrast to previous studies where lipid droplets have been shown to be protective against fatty acid-induced cell death, we showed that lipid droplets could not protect against GS-1-induced cytotoxicity. We also found a role for Fatty Acid Transport Protein 2 (FATP2) in the uptake of this compound. Collectively, this study demonstrates that GS-1 has effective pro-apoptotic antitumor activity in vitro and, together with the novel platform of fatty acid solubilization, contributes to the re-emerging field of fatty acids as potential anti-cancer therapeutics.

A supramolecular self-assembled nanomaterial for synergistic therapy of immunosuppressive tumor.

Triple negative breast cancer (TNBC) is an immunosuppressive "cold" tumor that lacks immune cell infiltration and activation, resulting in a poor response to immune checkpoint blockade (ICB) therapies. In addition, TNBC is poorly responsive to targeted therapies due to the absence of efficient molecular targets. A strategy that can block molecular signal transduction, stimulate immunogenicity, and activate the immune response is a promising approach to achieve ideal clinical benefit. Herein, we designed and synthesized an aggregation-induced emission luminogen (AIEgen)-conjugated self-assembling peptide that targets epidermal growth factor receptor (EGFR), named TPA-FFG-LA. TPA-FFG-LA peptides form nanoassemblies on the surface of EGFR-positive TNBC cells and are internalized into cells through endocytosis, which inhibit EGFR signaling transduction and provoke lysosomal membrane permeabilization (LMP). Upon light irradiation, the aggregated AIEgens produce massive reactive oxygen species (ROS) to exacerbate LMP and trigger immunogenic cell death (ICD), resulting in elimination of residual EGFR-negative tumor cells and exerting long-term antitumor effects. The in vitro and in vivo experiments verified that TPA-FFG-LA nanoassemblies suppress tumor growth, provoke immune cell activation and infiltration, and cause EGFR degradation and LMP. These results suggest that the combination of supramolecular assembly induced molecular targeting effects and lysosome dysfunction with ICD-stimulated immune activation is a plausible strategy for the efficient therapy of immunosuppressive TNBC.

An Activatable Near-Infrared Afterglow Theranostic Prodrug with Self-Sustainable Magnification Effect of Immunogenic Cell Death.

Herein, we report an activatable near-infrared (NIR) afterglow theranostic prodrug that circumvents high background noise interference caused by external light excitation. The prodrug can release hydroxycamptothecin (HCPT) in response to the high intratumoral peroxynitrite level associated with immunogenic cell death (ICD), and synchronously activate afterglow signal to monitor the drug release process and cold-to-hot tumor transformation. The prodrug itself is an ICD inducer achieved by photodynamic therapy (PDT). PDT initiates ICD and recruits first-arrived neutrophils to secrete peroxynitrite to trigger HCPT release. Intriguingly, we demonstrate that HCPT can significantly amplify PDT-mediated ICD process. The prodrug thus shows a self-sustainable ICD magnification effect by establishing an "ICD-HCPT release-amplified ICD" cycling loop. In vivo studies demonstrate that the prodrug can eradicate existing tumors and prevent further tumor recurrence through antitumor immune response.

Brush-like Polymer Prodrug with Aggregation-Induced Emission Features for Precise Intracellular Drug Tracking.

In this study, a brush-like polymer with aggregation-induced emission (AIE) features was synthesized for drug delivery and intracellular drug tracking. The polymer consisting of tetraphenylethene (TPE) chain-end as well as oligo-poly (ethylene glycol) (PEG) and hydrazine functionalities was successfully synthesized through copper (0)-mediated reversible-deactivation radical polymerization (Cu0-mediated RDRP). Anticancer drug doxorubicin (DOX) was conjugated to the polymer and formed a prodrug named TPE-PEGA-Hyd-DOX, which contains 11% DOX. The hydrazone between DOX and polymer backbone is a pH-sensitive linkage that can control the release of DOX in slightly acidic conditions, which can precisely control the DOX release rate. The drug release of 10% after 96 h in normal cell environments compared with about 40% after 24 h in cancer cell environments confirmed the influence of the hydrazone bond. The ratiometric design of fluorescent intensities with peaks at 410 nm (emission due to AIE feature of TPE) and 600 nm (emission due to ACQ feature of DOX) provides an excellent opportunity for this product as a precise intracellular drug tracker. Cancer cells confocal microscopy showed negligible DOX solution uptake, but an intense green emission originated from prodrug uptake. Moreover, a severe red emission in the DOX channel confirmed a promising level of drug release from the prodrug in the cytoplasm. The merged images of cancer cells confirmed the high performance of the TPE-PEGA-Hyd-DOX compound in the viewpoints of cellular uptake and drug release. This polymer prodrug successfully demonstrates low cytotoxicity in healthy cells and high performance in killing cancer cells.

Semiconducting Polymer Nanoparticles with Surface-Mimicking Protein Secondary Structure as Lysosome-Targeting Chimaeras for Self-Synergistic Cancer Immunotherapy.

Immunotherapy has received tremendous attention for tumor treatment, but the efficacy is greatly hindered by insufficient tumor-infiltration of immune cells and immunosuppressive tumor microenvironment. The strategy that can efficiently activate cytotoxic T lymphocytes and inhibit negative immune regulators will greatly amplify immunotherapy outcome, which is however very rare. Herein, a new kind of semiconducting polymer (SP) nanoparticles is developed, featured with surface-mimicking protein secondary structure (SPSS NPs) for self-synergistic cancer immunotherapy by combining immunogenic cell death (ICD) and immune checkpoint blockade therapy. The SPs with excellent photodynamic property are synthesized by rational fluorination, which can massively induce ICD. Additionally, the peptide antagonists are introduced and self-assembled into ß-sheet protein secondary structures on the photodynamic NP surface via preparation process optimization, which function as efficient lysosome-targeting chimaeras (LYTACs) to mediate the degradation of programmed cell death ligand-1 (PD-L1) in lysosome. In vivo experiments demonstrate that SPSS NPs can not only elicit strong antitumor immunity to suppress both primary tumor and distant tumor, but also evoke long-term immunological memory against tumor rechallenge. This work introduces a new kind of robust immunotherapy agents by combining well-designed photosensitizer-based ICD induction and protein secondary structures-mediated LYTAC-like multivalence PD-L1 blockade, rendering great promise for synergistic immunotherapy.

Measuring Cysteine Exposure in Unfolded Proteins with Tetraphenylethene Maleimide and its Analogs.

When proteostasis is challenged and becomes unbalanced, unfolded proteins can accumulate in the cells. Protein unfolding causes conformational changes and subsequent differentials in side-chain solvent accessibility and reactivity. In particular, when protein unfolds, non-disulfide-bonded cysteines that are usually buried in the native state can become surface exposed and thus accessible. A series of fluorogenic dyes including tetraphenylethene maleimide (TPE-MI) and its analogs were developed to capture cysteine exposure in unfolded proteins as a measure of unfolded protein load and proteostasis capacity in cells. These dyes are inherently non-fluorescent but show fluorescence turn-on effect when conjugated to unfolded proteins via reacting with exposed cysteines on the protein. Reacting with small biothiols such as glutathione does not induce fluorescence of these dyes. Here we describe the routine workflow to characterize unfolded proteins in vitro or unfolded proteomes in cells by TPE-MIs.

Red blood cell membrane-camouflaged nanoparticles loaded with AIEgen and Poly(I : C) for enhanced tumoral photodynamic-immunotherapy.

Red blood cell (RBC)-mimicking nanoparticles (NPs) offer a promising platform for drug delivery because of their prolonged circulation time, reduced immunogenicity and specific targeting ability. Herein, we report the design and preparation of RBC membrane-bound NPs (M@AP), for tumoral photodynamic-immunotherapy. The M@AP is formed by self-assembly of the positively charged aggregation-induced emission luminogen (AIEgen) (named P2-PPh3) and the negatively charged polyinosinic : polycytidylic acid (Poly(I : C)), followed by RBC membrane encapsulation. P2-PPh3 is an AIE-active conjugated polyelectrolyte with additional photosensitizing ability for photodynamic therapy (PDT), while Poly(I : C) serves as an immune-stimulant to stimulate both tumor and immune cells to activate immunity, and thus reduces tumor cell viability. When applied in tumor-bearing mice, the M@AP NPs are enriched in both the tumor region as a result of an enhanced permeability and retention (EPR) effect, and the spleen because of the homing effect of the RBC-mimicking shell. Upon light irradiation, P2-PPh3 promotes strong ROS generation in tumor cells, inducing the release of tumor antigens (TA). The anti-tumor immunity is further enhanced by the presence of Poly(I : C) in M@AP. Thus, this strategy combines the PDT properties of the AIE-active polyelectrolyte and immunotherapy properties of Poly(I : C) to achieve synergistic activation of the immune system for anti-tumor activity, providing a novel strategy for tumor treatment.

Construction of a Highly Sensitive Thiol-Reactive AIEgen-Peptide Conjugate for Monitoring Protein Unfolding and Aggregation in Cells.

Impairment of the protein quality control network leads to the accumulation of unfolded and aggregated proteins. Direct detection of unfolded protein accumulation in the cells may provide the possibility for early diagnosis of neurodegenerative diseases. Here a new platform based on a peptide-conjugated thiol-reactive aggregation-induced emission fluorogen (AIEgen), named MI-BTD-P (or D1), for labeling and tracking unfolded proteins in cells is reported. In vitro experiments with model proteins show that the non-fluorescent D1 only becomes highly fluorescent when reacted with the thiol group of free cysteine (Cys) residues on unfolded proteins but not glutathione or folded proteins with buried or surface exposed Cys. When the labeled unfolded proteins form aggregates, D1 fluorescence intensity is further increased, and fluorescence lifetime is prolonged. D1 is then used to measure unfolded protein loads in cells by flow cytometry and track the aggregate formation of the D1 labeled unfolded proteins using confocal microscopy. In combination with fluorescence lifetime imaging technique, the proteome at different folding statuses can be better differentiated, demonstrating the versatility of this new platform. The rational design of D1 demonstrates the outlook of incorporation of diverse functional groups to achieve maximal sensitivity and selectivity in biological samples.

In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe.

Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for ß-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with ß-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.

Recent advances in bioanalytical methods to measure proteome stability in cells.

Proteome stability constitutes an essential aspect of protein homeostasis (proteostasis). Proteostasis networks maintain proteins and their interactors in a defined conformation for their activity, localisation, and function. However, endogenous or exogenous stressors can perturb proteostasis integrity and deplete folding capacity, generating destabilized folding intermediates and deleterious aggregated species. Over the years, protein unfolding, misfolding and aggregation have been reported to be associated with aging and many diseases such as neurodegenerative diseases, diabetes, cardiac disease and toxicity, and cancers. Therefore, monitoring proteome stability is central to understanding underlying biological processes and mechanisms of disease progression. Herein, we review the recent bioanalytical methods to measure protein stability in cells on a proteome-wide scale.

Tumor-Triggered Disassembly of a Multiple-Agent-Therapy Probe for Efficient Cellular Internalization.

Integration of multiple agent therapy (MAT) into one probe is promising for improving therapeutic efficiency for cancer treatment. However, MAT probe, if entering the cell as a whole, may not be optimal for each therapeutic agent (with different physicochemical properties), to achieve their best performance, hindering strategy optimization. A peptide-conjugated-AIEgen (FC-PyTPA) is presented: upon loading with siRNA, it self-assembles into FCsiRNA -PyTPA. When approaching the region near tumor cells, FCsiRNA -PyTPA responds to extracellular MMP-2 and is cleaved into FCsiRNA and PyTPA. The former enters cells mainly by macropinocytosis and the latter is internalized into cells mainly through caveolae-mediated endocytosis. This two-part strategy greatly improves the internalization efficiency of each individual therapeutic agent. Inside the cell, self-assembly of nanofiber precursor F, gene interference of CsiRNA , and ROS production of PyTPA are activated to inhibit tumor growth.

Aggregation-Induced Emission Photosensitizers: From Molecular Design to Photodynamic Therapy.

Photodynamic therapy (PDT) has emerged as a promising noninvasive treatment option for cancers and other diseases. The key factor that determines the effectiveness of PDT is the photosensitizers (PSs). Upon light irradiation, the PSs would be activated, produce reactive oxygen species (ROS), and induce cell death. One of the challenges is that traditional PSs adopt a large flat disc-like structure, which tend to interact with the adjacent molecules through strong π-π stacking that reduces their ROS generation ability. Aggregation-induced emission (AIE) molecules with a twisted configuration to suppress strong intermolecular interactions represent a new class of PSs for image-guided PDT. In this Miniperspective, we summarize the recent progress on the design rationale of AIE-PSs and the strategies to achieve desirable theranostic applications in cancers. Subsequently, approaches of combining AIE-PS with other imaging and treatment modalities, challenges, and future directions are addressed.

Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Copper ions trigger disassembly of neurokinin B functional amyloid and inhibit de novo assembly.

The formation of amyloid is considered an intrinsic ability of most polypeptides. It is a structure adopted by many neuropeptides and neurohormones during the formation of dense core vesicles in secretory cells, yet the mechanisms mediating assembly and disassembly of these amyloids remain unclear. Neurokinin B is a neuropeptide thought to form an amyloid in secretory cells. It is known to coordinate copper, but the physiological significance of metal binding is not known. In this work we explored the amyloid formation of neurokinin B and the impact that metals had on the aggregation behaviour. We show that the production of neurokinin B amyloid is dependent on the phosphate concentration, the pH and the presence of a histidine at position 3 in the primary sequence. Copper(II) and nickel(II) coordination to the peptide, which requires the histidine imidazole group, completely inhibits amyloid formation, whereas zinc(II) slows, but does not inhibit fibrillogenesis. Furthermore, we show that copper(II) can rapidly disassemble preformed neurokinin B amyloid. This work identifies a role for copper in neurokinin B structure and reveals a mechanism for amyloid assembly and disassembly dependent on metal coordination.

Respiratory syncytial virus co-opts host mitochondrial function to favour infectious virus production.

Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV's impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV's unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.