Harnessing the Power of Sugar-Based Nanoparticles: A Drug-Free Approach to Enhance Immune Checkpoint Inhibition against Glioblastoma and Pancreatic Cancer.

Cancer cells have a high demand for sugars and express diverse carbohydrate receptors, offering opportunities to improve delivery with multivalent glycopolymer materials. However, effectively delivering glycopolymers to tumors while inhibiting cancer cell activity, altering cellular metabolism, and reversing tumor-associated macrophage (TAM) polarization to overcome immunosuppression remains a challenging area of research due to the lack of reagents capable of simultaneously achieving these objectives. Here, the glycopolymer-like condensed nanoparticle (∼60 nm) was developed by a one-pot carbonization reaction with a single precursor, promoting multivalent interactions for the galactose-related receptors of the M2 macrophage (TAM) and thereby regulating the STAT3/NF-κB pathways. The subsequently induced M2-to-M1 transition was increased with the condensed level of glycopolymer-like nanoparticles. We found that the activation of the glycopolymer-like condensed galactose (CG) nanoparticles influenced monocarboxylate transporter 4 (MCT-4) function, which caused inhibited lactate efflux (similar to inhibitor effects) from cancer cells. Upon internalization via galactose-related endocytosis, CG NPs induced cellular reactive oxygen species (ROS), leading to dual functionalities of cancer cell death and M2-to-M1 macrophage polarization, thereby reducing the tumor's acidic microenvironment and immunosuppression. Blocking the nanoparticle-MCT-4 interaction with antibodies reduced their toxicity in glioblastoma (GBM) and affected macrophage polarization. In orthotopic GBM and pancreatic cancer models, the nanoparticles remodeled the tumor microenvironment from "cold" to "hot", enhancing the efficacy of anti-PD-L1/anti-PD-1 therapy by promoting macrophage polarization and activating cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs). These findings suggest that glycopolymer-like nanoparticles hold promise as a galactose-elicited adjuvant for precise immunotherapy, particularly in targeting hard-to-treat cancers.

Engineered droplet-forming peptide as photocontrollable phase modulator for fused in sarcoma protein.

The assembly and disassembly of biomolecular condensates are crucial for the subcellular compartmentalization of biomolecules in the control of cellular reactions. Recently, a correlation has been discovered between the phase transition of condensates and their maturation (aggregation) process in diseases. Therefore, modulating the phase of condensates to unravel the roles of condensation has become a matter of interest. Here, we create a peptide-based phase modulator, JSF1, which forms droplets in the dark and transforms into amyloid-like fibrils upon photoinitiation, as evidenced by their distinctive nanomechanical and dynamic properties. JSF1 is found to effectively enhance the condensation of purified fused in sarcoma (FUS) protein and, upon light exposure, induce its fibrilization. We also use JSF1 to modulate the biophysical states of FUS condensates in live cells and elucidate the relationship between FUS phase transition and FUS proteinopathy, thereby shedding light on the effect of protein phase transition on cellular function and malfunction.

Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

Conformational switch of polyglutamine-expanded huntingtin into benign aggregates leads to neuroprotective effect.

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.

Ovarian Cancer Cell Adhesion/Migration Dynamics on Micro-Structured Laminin Gradients Fabricated by Multiphoton Excited Photochemistry.

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers.

The influence of pathological mutations and proline substitutions in TDP-43 glycine-rich peptides on its amyloid properties and cellular toxicity.

TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct ß-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308-310) during amyloidogenesis.

Inhibition of TDP-43 aggregation by nucleic acid binding.

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.

Signaling from membrane receptors to tumor suppressor WW domain-containing oxidoreductase.

The family of WW domain-containing proteins contains over 2000 members. The small WW domain module is responsible, in part, for protein/protein binding interactions and signaling. Many of these proteins are located at the membrane/cytoskeleton area, where they act as adaptors to receive signals from the cell surface. In this review, we provide molecular insights regarding recent novel findings on signaling from the cell surface toward WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1. More specifically, transforming growth factor beta 1 utilizes cell surface hyaluronidase Hyal-2 (hyaluronoglucosaminidase 2) as a cognate receptor for signaling with WWOX and Smad4 to control gene transcription, growth and death. Complement C1q alone, bypassing the activation of classical pathway, signals a novel event of apoptosis by inducing microvillus formation and WWOX activation. Deficiency in these signaling events appears to favorably support cancer growth.

Complement C1q activates tumor suppressor WWOX to induce apoptosis in prostate cancer cells.

BACKGROUND: Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. METHODOLOGY/PRINCIPAL FINDINGS: DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. CONCLUSIONS/SIGNIFICANCE: We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.