Amyloid-like Fibrils Research Articles

Modulation of hen egg white protein techno-functionality by amyloid formation

Hen egg white protein (EWP) is an ingredient in many food products. EWP containing amyloid-like fibrils (ALFs) can be prepared under food processing relevant conditions and have specific techno-functional properties which advantageously can be exploited in specific food products. Here, we investigated whether combinations of heating and NaCl addition or trypsin treatment improve EWP fibrillation and gel properties. Heating (75 °C, 150 min) control 0.2% EWP solutions resulted in limited fibrillation unless they also contained 150 mM NaCl, in which case increased levels of curly ALFs were observed. When similarly heated control EWP solutions were submitted to trypsin treatment (37 °C, 24 h, 150 rpm), both straight and curly ALFs were formed. Gels [5.0%–6.0% (w/v) EWP] prepared by heating (75 °C, 150 min) of 150 mM NaCl containing dispersions or by similar heating without NaCl addition but with trypsin treatment contained ALFs of similar morphology but shorter than those in control EWP solutions. Increased levels of curly or of both straight and curly ALFs in NaCl containing gels or gels prepared with trypsin treatment, respectively, resulted in higher EWP gel stiffness than when the EWP gels were prepared in water. While the presence of straight ALFs in trypsin-derived EWP gels resulted in fast gel breakdown, the presence of both curly and straight ALFs reduced the amount of EWP needed for gel formation. The level and morphology of EWP ALFs can thus be modulated by specific food relevant processing and the resultant ALFs have specific gelling properties.

Blueberry anthocyanins extract inhibits advanced glycation end-products (AGEs) production and AGEs-stimulated inflammation in RAW264.7 cells.

Pharmacological interference is considered to be a successful approach to inhibit advanced glycation end-products (AGEs) production and to block AGEs-induced diseases. Some synthetic medicines are effective for inhibiting the glycation reaction, but they cannot be widely applied in clinical as a result of their side effects and security concerns. The present study uses blueberry anthocyanins extract (BAE) to attenuate AGEs formation and AGEs-induced inflammatory response in vitro. In a bovine serum albumin-glucose model, BAE showed similar inhibitory activity on AGEs compared to the synthetic anti-glycation agent (aminoguanidine). The results showed that BAE exhibit strong anti-glycative action by scavenging glycosylated intermediates (Schiff base, fructosamine and α-dicarbonyl compounds), attenuating the molecular aggregation and amyloid-like fibrils formation, and preventing conformational modification. Additionally, BAE was found to dose-dependently inhibit the AGEs-induced secretions of nitric oxide and pro-inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α) in RAW264.7 cells. The anti-inflammation activity of BAE was mediated by down-regulating the expressions of critical inflammatory markers, inducible nitric oxide synthase and cyclooxygenase-2, through nuclear factor-kappa B signaling pathways inhibition. BAE could serve as a natural inhibitor for controlling AGEs and AGEs-induced chronic inflammation. © 2023 Society of Chemical Industry.

Improved coalescence and creaming stability of structured oil-in-water emulsions and emulsion gels containing ovalbumin amyloid-like fibrils produced by heat and enzymatic treatments

Proteins are well known stabilisers for emulsions, gels and foams. Hereby, the protein (supra)molecular structure may play an important role. In order to investigate that effect, 0.5% unheated (UH), heated (H) and combined heat and trypsin treated (H TT) ovalbumin (OVA) dispersions were evaluated for their capacity to stabilize 20–60% oil-in-water (O/W) emulsions. Such emulsions contained native OVA (UH OVA), amorphous and short fibrillar aggregates (H OVA) or long fibrils and peptides (H TT OVA). The experimental results indicated that emulsions containing UH OVA showed larger droplet sizes than those containing H OVA or H TT OVA and revealed a high extent of oiling-off. Although the emulsions containing H OVA contained smaller oil droplets, still a remarkable amount of oil separated upon centrifugation. In contrast, the H TT OVA stabilized emulsions did not display any oiling-off upon centrifugation, which was suggested to be due to the increased viscosity of the (gelled) H TT OVA dispersions used as continuous phase. Nevertheless, the viscosity of the latter emulsions was negatively affected by breakdown of the fibrillary (gel) structure during emulsification. When 60% O/W emulsions containing 1.6% H OVA were subjected to tryptic treatment only after emulsification, the emulsion gels were significantly stronger than the H TT OVA emulsions. Both emulsion types only showed the onset of creaming after several hours of centrifugation, whereas only the emulsions submitted to the enzymatic treatment after emulsification showed no evidence of coalescence. Overall, the experimental results show that emulsions containing long OVA fibrils, as obtained by trypsin treatment after heating, hold great promise for structured food applications.

Structural characteristics, component interactions and functional properties of gelatins from three fish skins extracted by five methods

Fish skin gelatin is an important functional product used in food, medicine and other industries. However, the structure and function of gelatins extracted with different methods differ significantly, thus limiting its production and application. This study used dry-salting, wet-salting, pepsin, acid and heat methods to extract gelatins from the skins of tilapia, grass carp and sea perch. Then, their structural characteristics (micro- and ultra-structure, amyloid-like fibril, etc.) and functional properties (viscosity, emulsifying performance, antioxidant abilities, etc.) were analyzed, and interaction between gelatin components were also explored. According to the results, the gelatins extracted with dry-salting and wet-salting methods had better reticular structure, larger fiber length/height, and higher viscosity properties, emulsifying and antioxidant capacity. The gelatin extracted by applying heat has the highest gel strength, and the gelatin extracted using pepsin had better thermal stability, water absorption capacity, and fat absorption capacity. Further analysis of component interaction showed that 11 types of collagens detected in the gelatins might promote the conversion of collagen to gelatin through self-assembly ability. The co-assembly of different types of collagens enhanced the properties of gelatin. Decorin had a positive effect on gelatin network structure, but Metallopeptidase inhibited the formation of network structure. Different methods can produce personalized gelatin products according to specific needs. The mining of component interaction would reveal the mechanism of gelatin formation and promote the development of gelatin synthetic biology.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111–142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111–142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111–142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111–142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111–142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Biomineralization in Barnacle Base Plate in Association with Adhesive Cement Protein.

Barnacles strongly attach to various underwater substrates by depositing and curing a proteinaceous cement that forms a permanent adhesive layer. The protein MrCP20 present within the calcareous base plate of the acorn barnacle Megabalanus rosa (M.rosa) was investigated for its role in regulating biomineralization and growth of the barnacle base plate, as well as the influence of the mineral on the protein structure and corresponding functional role. Calcium carbonate (CaCO3) growth on gold surfaces modified by 11-mercaptoundecanoic acid (MUA/Au) with or without the protein was followed using quartz crystal microbalance with dissipation monitoring (QCM-D), and the grown crystal polymorph was identified by Raman spectroscopy. It is found that MrCP20 either in solution or on the surface affects the kinetics of nucleation and growth of crystals and stabilizes the metastable vaterite polymorph of CaCO3. A comparative study of mass uptake calculated by applying the Sauerbrey equation to the QCM-D data and quantitative X-ray photoelectron spectroscopy determined that the final surface density of the crystals as well as the crystallization kinetics are influenced by MrCP20. In addition, polarization modulation infrared reflection-absorption spectroscopy of MrCP20 established that, during crystal growth, the content of β-sheet structures in MrCP20 increases, in line with the formation of amyloid-like fibrils. The results provide insights into the molecular mechanisms by which MrCP20 regulates the biomineralization of the barnacle base plate, while favoring fibril formation, which is advantageous for other functional roles such as adhesion and cohesion.

Dissolution of Lysozyme Amyloid Fibrils Using Magnetic Nanoparticles in an Alternating Magnetic Field: Design of an Effective Treatment for Cutaneous Amyloidosis

The purpose of this study was to apply functionalized magnetic nanoparticles for the treatment of amyloidosis, a disease characterized by the accumulation of aberrant protein forms with an insoluble amyloid structure. The dissolution and clearance of these extremely stable fibrils from lesions is very complicated. For this purpose, we examined the possibility of using magnetic nanoparticles that generate heat in an external alternating magnetic field with a frequency of 3.5 MHz. As a convenient model system, we used lysozyme fibrils. For the quantification of fibrillar status, we used Thioflavin T and Congo red, specific dyes which change their spectroscopic properties upon binding with the cross-beta structure of fibrils. We found that by using fluorescence, and polarization microscopy, as well as absorption spectrophotometry, the amyloid-like fibrils can be almost completely dissolved. The obtained results suggest that the application of magnetic nanoparticles could be a possible therapeutic intervention in cutaneous amyloidosis.

Cryo-EM structures of the D290V variant of the hnRNPA2 low complexity domain reveal the mechanism of its pathogenicity.

Human ribonucleoprotein hnRNPA2 belongs to the set of proteins that bind and shuttle RNA and also contain low-complexity domains (LCDs) having a limited repertoire of amino acid types. These LCDs aggregate into amyloid-like fibrils during the performance of their functions in phase separation. In previous work we determined the cryo-EM structure of the LCD of the wildtype hnRNPA2, finding an amyloid-like fibril that forms a reversible hydrogel. Here we determine the cryo-EM structures of three polymorphic amyloid fibrils formed by the recombinant D290V variant of hnRNPA2 LCD found in patients afflicted with MultiSystem Proteinopathy. In contrast to the wildtype, reversible fibrils, the variant fibrils are irreversible and more stable as judged by phase separation, thermal stability, and energetic calculations. Also, the PY-nuclear localization signal is masked in the fibril cores of all three variant polymorphs, thus rendering it inaccessible to chaperones for RNA trafficking and thereby interfering with function. These findings suggest how a single missense mutation converts functional amyloid-like fibrils to irreversible pathogenic amyloid.

Mechanism of action of beta-stranded antimicrobial (KL)n model peptides.

Here, we investigate model amphipathic β-stranded peptides, consisting of alternating lysine and leucine residues. These so called KLn peptides ([KL]n/2-NH2) show high antimicrobial activity against various bacterial strains. At the same time, they tend to aggregate into β-pleated amyloid-like fibrils. We systematically investigated their membrane affinity and their structural behaviour in the presence of lipid membranes as a function of peptide length, charge and hydrophobicity, using peptides with 6-26 amino acids. Different biophysical techniques (circular dichroism, solid-state NMR and fluorescence assays) were used and the results correlated with results of biological assays (antimicrobial and hemolytic assays), to identify possible mechanisms of action of KLn peptides. We find that the very short KL peptides are inactive, but longer peptides are highly hemolytic and aggregates strongly, especially in the presence of phosphate [1]. There is an optimal length of around 10 amino acids where the KL peptides have a high antimicrobial effect and low hemolysis. At this length, peptides are long enough to form stable β-stranded structure but flexible enough to not aggregate very fast in solution. A similar length behaviour has been observed for other similar peptides made from alternating hydrophobic and charged residues [2]. Vesicle leakage is length-dependent and peptides need at least 12 amino acids to induce high leakage. This threshold length does not depend on membrane thickness. This indicates that pores are probably not formed by these peptides, and this was further confirmed by solid-state 15N- and 19F-NMR experiments, whose results were incompatible with pore models [3]. Thus, it seems that the peptides are bound to the membrane surface and destabilize the membrane by some kind of carpet mechanism.

Development and mechanical properties of soy protein fibrils-chitin nanowhiskers complex gel

To enrich the use of protein-polysaccharide complex gel as texture assistance, we introduced a soy protein fibrils-chitin nanowhiskers complex gel system. Firstly, a stable positively charged chitin nanowhiskers (CNW) suspension and amyloid-like soy protein fibrils (SPF) were fabricated. We found that the self-assembled SPF and CNW formed complex with each other at pH 5.0–8.0 due to attractive interactions caused by their carrying opposite electrical charges. Then, the MTGase-induced SPF-CNW complex gel were formed and investigated by instrumental analysis. The results showed that the presence of CNW significantly increased the β-Sheet structure in complex gel and the rheological modules of complex gel were pH-dependent and showing G' > G" at all pHs. The SPF-CNW complex gel showed a strong and elastic textural property during compression test at pH 6.0. Meanwhile, the frequency scan showed that the SPF-CNW system is less dependent on the frequency and belonged to the typical physical gel. According to the results of SEM and CLSM images, the SPF, CNW and complex gel presented spherical shape, rod shape and linear alignment microstructure, respectively. We inferred that the CNW provided a framework for gel formation and introduced rigidity to the SPF gel. Thanks to the attractive interactions, the SPF molecules were attached to the growth of CNW to form a longer fiber structure. After the gelation process, the aggregates further interacted with each other and finally form complex gel with linear, ordered and compact microstructure.

Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation.

Mammary tumor-associated amyloidosis (MTAA) in dogs is characterized by amyloid deposition in the stroma of mammary adenoma or carcinoma; however, the amyloid precursor protein remains unknown. We attempted to identify an amyloid precursor protein and elucidated its etiology by characterizing 5 cases of canine MTAA. Proteomic analyses of amyloid extracts from formalin-fixed paraffin-embedded specimens revealed α-S1-casein (CASA1) as a prime candidate and showed the N-terminal truncation of canine CASA1. Both immunohistochemistry and immunoelectron microscopy showed that amyloid deposits or fibrils in MTAA cases were positive for CASA1. Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction revealed the complete mRNA sequence encoding CASA1, whose expression was significantly higher in the amyloid-positive group. The recombinant protein of the N-terminal-truncated canine CASA1 and the synthetic peptides derived from canine and human CASA1 formed amyloid-like fibrils in vitro. Structural prediction suggested that the N-terminal region of CASA1 was disordered. Previously, full-length CASA1 was reported to inhibit the amyloidogenesis of other proteins; however, we demonstrated that CASA1 acquires amyloidogenicity via excessive synthesis followed by truncation of its disordered N-terminal region. By identifying a novel in vivo amyloidogenic protein in animals and revealing key mechanistic details of its associated pathology, this study provides valuable insights into the integrated understanding of related proteopathies.

Modification of ovalbumin by the enzymatic method: Consequences for foaming characteristics of fibrils

Amyloid-like fibrils from proteins possess unique functional properties for food and many other uses. This study reports the effect of different trypsin concentrations (0.1%, 0.2%, 1.0%) on the formation and foaming properties of ovalbumin protein amyloid fibrils. The fibrils were characterized through Thioflavin T (ThT) fluorescence, TEM, Circular dichroism spectroscopy, viscosity, particle size. The ThT results revealed that the moderate modification could promote protein unfolding, consequently facilitating fibril formation. Based on the results of TEM, moderate modification promoted the elongation of fibrils, and the formation of worm-like fibrils was shown. Furthermore, the structure of modified fibril aggregates was compact, which highly promoted the foaming ability and stability. According to these findings, fibrils formed by enzyme-modified OVA is a promising method that can expand the application of foaming agents and stabilizers in the food industry.

Comparison of the assembly behavior and structural characteristics of arachin and conarachin amyloid-like fibrils

In this study, the assembly behavior and structural characteristics of arachin (Ara) and conarachin (Con) amyloid-like fibrils formed by heating at 80 °C and 95 °C under acidic conditions were investigated. The results showed that Ara exhibited higher Thioflavin T fluorescence intensity during the fibrillation process and assembled into longer amyloid-like fibrils compared to Con, indicating that Ara had a stronger ability to form amyloid-like fibrils. Ara and Con were hydrolyzed into small peptides during the initial heating process, leading to decreased average particle sizes, as evidenced by the degraded subunits. Then the average particle size increased with the small peptides assembling into ordered amyloid-like fibrils with high content of β-sheets. Based on the results of surface hydrophobicity and the content of free –SH, the hydrophobic interactions played a more important role in the fibrillation process of Ara, while more surface free –SH involved in the formation of Con fibrils. Higher temperature (95 °C) accelerated the hydrolysis of proteins and subsequent self-assembly process. Furthermore, these amyloid-like fibrils had no in vitro cytotoxicity, showing the potential application in future food production. In summary, this work provides us a deep understanding of the formation mechanism and structural characteristics of Ara and Con amyloid-like fibrils.

OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

RIPK3–ZBP1–MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2′-5′ oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3–ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein–protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3–ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

Amyloid-Based Albumin Hydrogels.

Amyloid fibrils may serve as building blocks for the preparation of novel hydrogel materials from abundant, low-cost, and biocompatible polypeptides. This work presents the formation of physically cross-linked, self-healing hydrogels based on bovine serum albumin at room temperature through a straightforward disulfide reduction step induced by tris (2-carboxyethyl) phosphine hydrochloride. The structure and surface charge of the amyloid-like fibrils is determined by the pH of the solution during self-assembly, giving rise to hydrogels with distinct physicochemical properties. The hydrogel surface can be readily functionalized with the extracellular matrix protein fibronectin and supports cell adhesion, spreading, and long-term culture. This study offers a simple, versatile, and inexpensive method to prepare amyloid-based albumin hydrogels with potential applications in the biomedical field.

Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Peptide from NSP7 is able to form amyloid-like fibrils: Artifact or challenge to drug design?

•We found potential amyloidogenic fragment in NSP7 SARS-CoV2 protein in silico•NSP7 (52–62) fragment is able to form amyloid-like fibrils•The possibility of using such a peptide as the basis for an antiviral drug is discussed

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

Amyloid-like aggregation influenced by lead(II) and cadmium(II) ions in hen egg white ovalbumin

The aggregation of proteins into fibrillar, amyloid-like aggregates generally results in an improved, positive effect on various techno-functional properties within food products, such as gelation, emulsification, and foam stabilization. These highly stable structures, characterized by their repetitive, β-sheet rich motifs, may develop as the result of the thermal treatment of protein-rich food products. Heavy metal ions can influence amyloid-like aggregation of food proteins. Lead(II) and cadmium(II) represent some of the most abundant and common environmental water and food pollutants.In this work, the influence of heavy metal ions, lead and cadmium on amyloid-like aggregation of ovalbumin at high temperatures (90 °C) and under acidic conditions (pH 2.0) was investigated. Ovalbumin is used as a general model for how heavy metals can affect amyloid-like aggregation of a food protein. Structural changes were monitored via Thioflavin T and 8-Anilino-1-naphthalenesulfonic acid fluorescence, Fourier-Transform infrared spectroscopy, atomic force microscopy, dynamic light scattering, as well as computational analyses. The obtained results indicate that the added heavy metal ions bind to different sites within ovalbumin prior to thermal treatment. Lead binding sites are closer to the hydrophobic regions of an protein, while cadmium ion binding sites are more exposed. This specific binding of metal ions affects the morphologies of amyloid-like aggregates, resulting in lead-induced branching of amyloid-like fibrils, or cadmium-induced tangling of fibrils into dense amyloid clusters. This additive effect of heavy metal ions is most evident in ovalbumin samples which contain a mixture of both heavy metal ions.

RF04 | PSUN233 Design of an Ultra-Stable Insulin/Glucagon Fusion Protein

Abstract Insulin replacement therapy is lifesaving for insulin-dependent diabetic patients, but it comes with an ever-present threat of hypoglycemia, which can eventually cause ischemic events, arrhythmias, neurological damage, coma, or even death. Consequently, the risk of hypoglycemia remains the most significant obstacle that prevents insulin-dependent patients from attaining adequate metabolic control. One approach to solve this issue involves the co-injection of insulin and glucagon at a fixed ratio, which has been shown to improve glycemic control with prevention of hypoglycemia. The co-administration of both hormones is based on two known physiological processes: first, insulin is a potent inhibitor of glucagon activity, especially under hyperglycemic conditions, and second, glucagon overpowers insulin and stimulates hepatic glucose production under hypoglycemic circumstances. In this work, we took this concept further into developing an insulin/glucagon fusion protein that will provide glycemic control (by the insulin portion) while preventing hypoglycemia (through the glucagon portion). Because both hormones are known to be unstable and form amyloid-like fibrils, our approach consists of using a fibrillation-resistant single-chain insulin (SCI) analog fused to a novel fibrillation-resistant glucagon analog. These molecules were independently synthesized by Solid-Phase Peptide Synthesis (SPPS) and linked together from the C-Terminus of the glucagon analog to the N-Terminus of the SCI analog by trypsin-mediated ligation. By themselves or when fused, these ultra-stable analogs didn't form fibrils after one month of agitation at 37°C in PBS (pH 7.4). By contrast, native insulin started fibrillation at 12.14 ± 3.65 hours, and native glucagon at time 0. The glucagon analog was designed to be less potent than the native molecule to allow unopposed insulin activity while in hyperglycemia but maintaining full agonism to prevent hypoglycemia. The in vitro glucagon activity was measured by cAMP production in HEK293 expressing the glucagon receptor. The glucagon analog was determined to be a full agonist with an EC50 of 125.7 nM, a ∼58-fold potency difference to the native molecule (2.175 nM). The fusion protein was tested in vitro and in vivo to determine the activities of both moieties when fused. The in vitro insulin activity of the fusion protein measured by traditional and in-cell western blot was comparable to the SCI alone. The in vitro glucagon activity of the fusion protein was also similar to the glucagon analog alone (EC50 151.2 nM), therefore validating our fusion approach. The in vivo insulin and glucagon activities are currently being tested by subcutaneous injection in diabetic (STZ) and normal rats. So far, both insulin and glucagon moieties have shown to be fully active, and hypoglycemia has not been observed in rats. Increasing doses studies are underway. We believe that this approach will allow the design of next-generation insulin treatments protected against hypoglycemia, ultra-stable, and amenable for multiplatform delivery. Presentation: Saturday, June 11, 2022 1:54 p.m. - 1:59 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.