Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Natural pigment from Monascus: The production and therapeutic significance.

OBJECTIVE: The present review highlights the advantages of using natural colorant over the synthetic one. We have discussed the fermentation parameters that can enhance the productivity of Monascus pigment on agricultural wastes. BACKGROUND: Food industry is looking for natural colours because these can enhance the esthetic value, attractiveness, and acceptability of food while remaining nontoxic. Many synthetic food colours (Azorubine Carmoisine, quinoline) have been prohibited due to their toxicity and carcinogenicity. Increasing consumer awareness towards the food safety has forced the manufacturing industries to look for suitable alternatives. In addition to safety, natural colorants have been found to have nutritional and therapeutic significance. Among the natural colorants, microbial pigments can be considered as a viable option because of scalability, easier production, no seasonal dependence, cheaper raw materials and easier extraction. Fungi such as Monascus have a long history of safety and therefore can be used for production of biopigments. METHOD: The present review summarizes the predicted biosynthetic pathways and pigment gene clusters in Monascus purpureus. RESULTS: The challenges faced during the pilot-scale production of Monascus biopigment and taming it by us of low-cost agro-industrial substrates for solid state fermentation has been suggested. CONCLUSION: Keeping in mind, therapeutic properties of Monascus pigments and their derivatives, they have huge potential for industrial and pharmaceutical application. APPLICATION: Though the natural pigments have wide scope in the food industry. However, stabilization of pigment is the greatest challenge and attempts are being made to overcome this by complexion with hydrocolloids or metals and by microencapsulation.

Self-assembly of stimuli-responsive coiled-coil fibrous hydrogels.

Owing to their tunable properties, hydrogels comprised of stimuli-sensitive polymers are one of the most appealing scaffolds with applications in tissue engineering, drug delivery and other biomedical fields. We previously reported a thermoresponsive hydrogel formed using a coiled-coil protein, Q. Here, we expand our studies to identify the gelation of Q protein at distinct pH conditions, creating a protein hydrogel system that is sensitive to temperature and pH. Through secondary structure analysis, transmission electron microscopy, and rheology, we observed that Q self-assembles and forms fiber-based hydrogels exhibiting upper critical solution temperature behavior with increased elastic properties at pH 7.4 and pH 10. At pH 6, however, Q forms polydisperse nanoparticles, which do not further self-assemble and undergo gelation. The high net positive charge of Q at pH 6 creates significant electrostatic repulsion, preventing its gelation. This study will potentially guide the development of novel scaffolds and functional biomaterials that are sensitive towards biologically relevant stimuli.

Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.

Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities.

Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.

Efficient Dual siRNA and Drug Delivery Using Engineered Lipoproteoplexes.

An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (∼40 µg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.

Intratumoral IFN-γ or topical TLR7 agonist promotes infiltration of melanoma metastases by T lymphocytes expanded in the blood after cancer vaccine.

BACKGROUND: Immune-mediated melanoma regression relies on melanoma-reactive T cells infiltrating tumor. Cancer vaccines increase circulating melanoma-reactive T cells, but little is known about vaccine-induced circulating lymphocytes (viCLs) homing to tumor or whether interventions are needed to enhance infiltration. We hypothesized that viCLs infiltrate melanoma metastases, and intratumoral interferon (IFN)-γ or Toll-like receptor 7 (TLR7) agonism enhances infiltration. METHODS: Patients on two clinical trials (Mel51 (NCT00977145), Mel53 (NCT01264731)) received vaccines containing 12 class I major histocompatibility complex-restricted melanoma peptides (12MP). In Mel51, tumor was injected with IFN-γ on day 22, and biopsied on days 1, 22, and 24. In Mel53, dermal metastases were treated with topical imiquimod, a TLR7 agonist, for 12 weeks, and biopsied on days 1, 22, and 43. For patients with circulating T-cell responses to 12MP by IFN-γ ELISpot assays, DNA was extracted from peripheral blood mononuclear cells (PBMCs) pre-vaccination and at peak T-cell response, and from tumor biopsies, which underwent T-cell receptor sequencing. This enabled identification of clonotypes induced in PBMCs post-vaccination (viCLs) and present in tumor post-vaccination, but not pre-vaccination. RESULTS: Six patients with T-cell responses post-vaccination (Mel51 n = 4, Mel53 n = 2) were evaluated for viCLs and vaccine-induced tumor infiltrating lymphocytes (viTILs). All six patients had viCLs, five of whom were evaluable for viTILs in tumor post-vaccination alone. Mel51 patients had viTILs identified in day 22 tumors, post-vaccination and before IFN-γ (median = 2, range = 0-24). This increased in day 24 tumors after IFN-γ (median = 30, range = 4-74). Mel53 patients had viTILs identified in day 22 tumors, post-vaccination plus imiquimod (median = 33, range = 2-64). Three of five evaluable patients across both trials had viTILs with vaccination alone. All five had enhancement of viTILs with tumor-directed therapy. viTILs represented 0.0-2.9% of total T cells after vaccination alone, which increased to 0.6-8.7% after tumor-directed therapy. CONCLUSION: Cancer vaccines induce expansion of new viCLs, which infiltrate melanoma metastases in some patients. Our findings identify opportunities to combine vaccines with tumor-directed therapies to enhance T-cell infiltration and T cell-mediated tumor control. These combinations hold promise in improving the therapeutic efficacy of antigen-specific therapies for solid malignancies.

Tertiary lymphoid structures in desmoplastic melanoma have increased lymphocyte density, lymphocyte proliferation, and immune cross talk with tumor when compared to non-desmoplastic melanomas.

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8+ T-cell and CD20+ B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM.

Antioxidative, anti-inflammatory, and anticancer properties of the red biopigment extract from Monascus purpureus (MTCC 369).

In this study, the Monascus purpureus (MTCC 369) extracted biopigment produced by solid-state fermentation was evaluated for its therapeutic potential using human prostate LNCaP cells. Antioxidant efficacy of the red biopigment determined using 2,2 diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, and ferric reducing antioxidant power assays was found to be 53.16%, 86.27%, and 13.83%, respectively. In addition, expression studies of target gene superoxide dismutase 2 (SOD-2) showed that increasing concentrations (10-50 µg/ml) of the biopigment enhanced its expression from 0.91- to 1.905-fold. An inhibitory effect of 0.424-0.627-fold was observed in the expression of glutathione peroxidase (GPX) with a similar increase in biopigment concentration. Addition of quercetin (positive control) at 50 µg/ml led to 0.295-fold decrease in GPX expression. In contrast, the expression of SOD-2 increased by 1.026-fold in the presence of quercetin. The biopigment also showed an increased serological IL-10 expression (an anti-inflammatory agent) ranging from 1034.58 to 4657.89 pg/ml. Treatment of LNCaP cells with the red biopigment (10-100 µg/ml) resulted in significant (p < .05) reduction (upto 79.86%) in viability and 51.79%-89.86% reduction in cell metabolic activity. Fluorescent microscopy examination of red biopigment-treated cells showed significant inhibition of normal cellular morphology including condensed nuclei, membrane blebbing, and apoptotic bodies, thus confirming its cytotoxic potential. Results of this study revealed that the red biopigment has the potential to modulate the expression of antioxidative and anti-inflammatory markers in addition to being cytotoxic to the LNCaP cancer cells. PRACTICAL APPLICATIONS: These findings indicate that cell treatment with red biopigment has the potential to modulate anti-oxidative, pro-inflammatory and anti-inflammatory genes for therapeutic effects, which is further enhanced by its cytotoxic activity against cancer cells. Considering these cell-based observations, Monascus red biopigment has ample potential as a useful supplement to formulate therapeutic products that delay the development of inflammatory-related diseases and associated complications.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Enhancing organophosphate hydrolase efficacy via protein engineering and immobilization strategies.

Organophosphorus compounds (OPs), developed as pesticides and chemical warfare agents, are extremely toxic chemicals that pose a public health risk. Of the different detoxification strategies, organophosphate-hydrolyzing enzymes have attracted much attention, providing a potential route for detoxifying those exposed to OPs. Phosphotriesterase (PTE), also known as organophosphate hydrolase (OPH), is one such enzyme that has been extensively studied as a catalytic bioscavenger. In this review, we will discuss the protein engineering of PTE aimed toward improving the activity and stability of the enzyme. In order to make enzyme utilization in OP detoxification more favorable, enzyme immobilization provides an effective means to increase enzyme activity and stability. Here, we present several such strategies that enhance the storage and operational stability of PTE/OPH.

Recent trends in peptide and protein-based hydrogels.

Hydrogels are classic examples of biomaterials that have found its niche in biomedical and allied fields. Here, we describe examples of peptide-based and protein-based hydrogels with a focus on smart gels that respond to various stimuli including temperature, pH, light, and ionic strength. With the recent advancements in computational modeling, it has been possible to predict as well as design peptide and protein sequences that can assemble into hydrogels with unique and improved properties. We briefly discuss coarse grained and atomistic simulations in designing peptides that can form hydrogels. In addition, we highlight the trends that will influence the future design and applications of hydrogels, with emphasis on bioadhesion, exosomes delivery, tissue and organoids engineering, and even intracellular production of gels.

Self-Assembled Protein- and Peptide-Based Nanomaterials.

Considerable effort has been devoted to generating novel protein- and peptide-based nanomaterials with their applications in a wide range of fields. Specifically, the unique property of proteins to self-assemble has been utilized to create a variety of nanoassemblies, which offer significant possibilities for next-generation biomaterials. In this minireview, we describe self-assembled protein- and peptide-based nanomaterials with focus on nanofibers and nanoparticles. Their applications in delivering therapeutic drugs and genes are discussed.

Protein-Engineered Functional Materials.

Proteins are versatile macromolecules that can perform a variety of functions. In the past three decades, they have been commonly used as building blocks to generate a range of biomaterials. Owing to their flexibility, proteins can either be used alone or in combination with other functional molecules. Advances in synthetic and chemical biology have enabled new protein fusions as well as the integration of new functional groups leading to biomaterials with emergent properties. This review discusses protein-engineered materials from the perspectives of domain-based designs as well as physical and chemical approaches for crosslinked materials, with special emphasis on the creation of hydrogels. Engineered proteins that organize or template metal ions, bear noncanonical amino acids (NCAAs), and their potential applications, are also reviewed.

Protein-Engineered Nanoscale Micelles for Dynamic 19F Magnetic Resonance and Therapeutic Drug Delivery.

Engineered proteins provide an interesting template for designing fluorine-19 (19F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed "fluorinated thermoresponsive assembled protein" (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting 19F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation ( T2) times and unaffected spin-lattice relaxation ( T1) times. The nearly unchanging T1 relaxation rates and linearly dependent T2 relaxation rates have allowed for detection via zero echo time 19F MRI, and the in vivo MR potential has been preliminarily explored using 19F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive 19F MRI/MRS-traceable theranostic agents.

Optimization of gluco-amylase production from Aspergillus spp. for its use in saccharification of liquefied corn starch.

Fungal gluco-amylase is required for the production of sugars from starchy substrates. Commercially available fungal gluco-amylase is quite costly which makes the process uneconomical. This study was undertaken to standardize physico-chemical parameters for optimum production of gluco-amylases from Aspergillus spp. Two fungal cultures, i.e., Aspergillus niger and Aspergillus terreus, were compared for gluco-amylase activity both under stationary and shake flask conditions. Among two fungal cultures, maximum gluco-amylase activity was shown by A. niger (243.09 U/ml) under stationary conditions as compared to A. terreus (126.34 U/ml). Gluco-amylase activity of A. niger increases by 42.48% from 243.09 to 346.35 U/ml after optimization using response surface methodology, whereby a substrate concentration of 7%, yeast extract 0.25%, temperature 32.5 °C and pH 5.5 were found to be optimum for gluco-amylase production. Crude enzyme was compared with commercial enzyme and it was found that when 500 U of Glucoamylase ex. Rhizopus were inoculated into starch-supplemented minimal media (SSMM) liquefied using 2 g of fungal diastase, it increases the reducing sugar concentration from 2.19 to 21.15 mg/ml and a saccharification efficiency of 77.7% was achieved, whereas 1.5 ml of crude enzyme (extracted from A. niger) was able to produce 14.46 mg/ml of reducing sugars with a saccharification efficiency of 53.2%.

Design and Characterization of Fibers and Bionanocomposites Using the Coiled-Coil Domain of Cartilage Oligomeric Matrix Protein.

Tremendous effort has been dedicated to the design and assembly of bioinspired protein-based architectures with potential applications in drug delivery, tissue engineering, biosensing, and bioimaging. Here, we describe our strategy to generate fibers and bionanocomposites using the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Our construct, Q, engineered by swapping particular regions of COMPcc to optimize surface charge, self-assembles to form nanofibers. The Q protein nanofibers can efficiently bind curcumin to form robust mesofibers that can be potentially used for drug delivery and biomedical applications. In addition, using the same Q protein, we describe the biotemplation of gold nanoparticles (AuNP) in the presence and absence of the hexahistidine tag (His-tag). The Q bearing His-tag·AuNP (Q·AuNP) readily deposits on electrode surfaces, while Q without His-tag·AuNP (Qx·AuNP) stabilizes the soluble protein·gold bionanocomposites for several days without aggregating.

Binding and backbone dynamics of protein under topological constraint: calmodulin as a model system.

Herein we present the effect of artificially imposed topological constraint on calmodulin (CaM) backbone dynamics and its molecular recognition behavior. While backbone dynamics of CaM remain largely unperturbed, the thermodynamic profile of CaM binding to the smooth-muscle myosin light-chain kinase (smMLCK) peptide is modulated significantly.

Expression of Cellulolytic Enzyme as a Fusion Protein That Reacts Specifically With a Polymeric Scaffold.

The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes built onto a common protein scaffold to facilitate the breakdown of the polymeric substrate, cellulose. One strategy for mimicking these highly effective nanomachines may involve the use of synthetic scaffolds that can react to and organize multiple engineered enzymes to promote synergistic action between the enzymes on the scaffold. As an example of the earlier strategy, we describe here an approach for the expression of cellulolytic enzymes with a serine esterase tag, and the rapid reaction between the tag and the end-functionalized polymers to form enzyme-polymer-enzyme multienzyme conjugates. In principle, this general and versatile supramolecular approach may be used to organize specific cellulolytic enzymes onto synthetic scaffolds to form multienzyme complexes to potentially work in synergy for enhanced biological activities. Best reaction conditions, good activities of the armored cellulolytic enzymes and the design of optimal protein linker in the fusion protein are discussed in detail. If other reactive tags are included on the enzyme in future, multiple types of synergistic enzymes may be positioned at specific sites on a designed polymer scaffold that mimics the complex structure and enhanced function of natural cellulosomes. This type of nanoarmoring of multiple enzymes on a nanoscale might also enhance enzyme stability, when compared to the unprotected enzymes.

Suppression of inflammatory and allergic responses by pharmacologically potent fungus Ganoderma lucidum.

Acute inflammation is the result of a complex signal transduction pathway that protects and heals our body and is necessary for our good health and normal wellbeing. Whereas, chronic inflammation can be correlated well with the onset of a plethora of autoimmune disorders; rheumatoid arthritis, systemic lupus and polymyalgia, rheumatic and other diseases like asthma, inflammatory bowel diseases, cardiovascular disorders, ulcerative colitis and Crohn's disease. Also, it has been reported to be associated with the onset of various cancers. An effective anti-inflammatory drug should be able to inhibit the development of chronic inflammation without interfering in normal homeostasis. A number of herbal drugs have been identified in the past that can target inflammatory cytokines. Among these, Ganoderma lucidum: a powerful medicinal mushroom has been found to possess immune-modulating and immune-potentiating capabilities and has been characterized as a wonder herb. This review mainly focuses on the molecular mechanism of anti-inflammatory and antiallergic action of this mushroom and also sheds light on various patent studies related to its pharmacological action.