In Alzheimer’s disease, neurons undergo acidosis as their pH drops from ∼7.1 to ∼6.5. Here we elucidate the molecular mechanism of specific Tau aggregation only at this lower pH. We show that a specific phosphorylation event in the Tau R4 domain is coupled to this pH drop to drive a defined phase transition from condensates to fibrils. Using a combination of experimental and computational studies, our results demonstrate that site-specific phosphorylation of Ser352 is the molecular switch that induces aggregation of Tau only at the more acidic, disease-related pH. We designed and synthesized a phosphopeptide library covering all phosphorylation patterns of the Tau-R4 domain and tested its response to controlled acidification using an array of complementary biophysical methods. This revealed that only a single phosphorylation of Ser352 led to acid-driven aggregation of Tau-R4. At neutral pH, Tau-R4 with a phosphorylation on Ser352 formed liquid condensates. These condensates converted irreversibly into amyloid filaments as the pH gradually decreased towards the level associated with pathological acidosis. Using a combination of ³¹P-and ¹H - NMR, MD simulations and microscopy studies, we show that the mechanism by which the phosphorylation of Ser352 Tau R4 exerts its effects is based on the unique position of this residue within the protein structure. pSer352 is located at the inside of the tip of a β-hairpin, pointing into the hydrophobic core of the amyloid fold. This buried position increases its effective pK□, resulting in compacting of the hairpin following pSer352 protonation upon acidification. This shortens the inter-sheet distance at this position and tightens the filament. We conclude that this single protonation event of the pSer352 phosphate is responsible for the macroscopic phase transition from condensates to aggregates. Our results provide the molecular explanation for the specific aggregation of Tau under the pathologically relevant acidic pH, which is substantially different from its behavior at neutral pH.

Protein phosphorylation is one of the most common and versatile regulatory mechanisms in cells. Most human proteins are phosphorylated at multiple sites, giving rise to large numbers of possible phosphorylation patterns. Each phosphorylation pattern can lead to a different functional or pathological outcome. Yet, linking defined phosphorylation patterns to specific biological functions remains a major experimental challenge. In this review we describe the main strategies to study phosphorylation patterns at the protein and domain levels and highlight how they complement each other. We first discuss cellular approaches, including phosphomimetics, kinase-based assays, and genetic code expansion, which allow working in a native environment but have their significant drawbacks. We then describe in vitro methods, such as enzymatic phosphorylation and semi-synthetic phosphoproteins generated by ligation, which afford mechanistic insights but result in low yields and are difficult to scale for producing libraries. We focus on synthetic phosphopeptide libraries as tools that offer precise control over the number and position of phosphosites and are uniquely suited for systematic mapping of phosphorylation patterns. This comes at a price of not working at the protein level, but rather at the domain level. Peptide libraries are often used for preliminary identification of key phosphorylations, later studied in detail at the protein level. We conclude that ideally more than one method should be used and that these methods should not be viewed as competing but rather as complementary. A combined use of several of these approaches provides a practical toolbox for dissecting how phosphorylation patterns regulate protein behavior.

Cultivated meat (CM) is an emerging field of research that applies cell-culture and bioprocessing technologies to the production of animal-derived food products without conventional animal farming. Growing global demand for meat, coupled with environmental and resource constraints associated with existing production systems, has motivated increasing academic, industrial, and policy interest in CM. Beyond its research and commercial implications, CM has the potential to contribute to social stability and food security, particularly in regions with limited arable land and high dependence on food imports, and may reduce vulnerability to global disruptions such as zoonotic disease outbreaks (e.g., avian influenza). This review offers a comprehensive analysis of the field, addressing the growing demand for CM, technical advancements, and the challenges in translating CM production into scalable applications. It examines current technological obstacles, highlights recent research progress, and explores potential solutions for achieving sustainable growth in the industry. By evaluating the intricacies of CM, this review aims to provide insights into the strategies needed to advance this innovative field and meet future demands for sustainable and secure food sources.

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerativediseases, including Alzheimer’s disease (AD). The molecular processes driving aggre-gation in disease are still being uncovered, highlighting the need for innovative toolsto study aggregation reactions. Here, we introduce FibrilPaint1 as a tool to measurethe size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils.FibrilPaint1 is a 22mer peptide with exciting properties: i) FibrilPaint1 binds fibrilswith nanomolar affinity; ii) it also binds to precursors, down to a size of only 4 layers;iii) it does not bind to monomers; iv) it is fluorescently labeled, which allows monitor-ing and localizing interactions; v) FibrilPaint1 recognizes various Tau fibrils, includingpatient- derived fibrils from AD, corticobasal degeneration (CBD), and frontotemporaldementia (FTD); vi) it also binds to fibrils from amyloids derived from Amyloid- β,α- synuclein, and huntingtin vii) FibrilPaint1 is selective for the amyloid state and doesnot have background binding to amorphous aggregates, blood serum, or cell lysate. Incombination with flow- induced dispersion analysis (FIDA), a microfluidics technology,we determined the molecular size of amyloid fibrils with submicroliter sample volumes.This setup acts as a molecular ruler at layer resolution—we determined Tau fibril lengthfrom 4 to 1100 layers in solution. This is an interesting parameter for molecular studiesin dementia, with potential for diagnostic applications.

Ovarian cancer is the most fatal cancer of female reproductive organs. Ovarian cancer is typically diagnosed at a late stage, after metastasis occurred, leading to a 5-years relative survival rate of only ∼5%. Here, we demonstrate the anti-ovarian cancer properties of a peptide derived from the human protein CISD2/NAF-1 (3D-NAF-1^44-67-6K). This peptide selectively permeates the plasma membrane of ovarian cancer SKOV-3 cells without affecting healthy cells. 3D-NAF-1^44-67-6K targets and destroys the cancer cells’ mitochondria which leads to cancer cell death. In vivo studies of mice carrying xenograft tumours of SKOV-3 showed that the peptide significantly decreased the overall size and growth rate of both primary and metastatic ovarian cancer tumours. We further show that 3D-NAF-1^44-67-6K has a broad-spectrum anticancer activity targeting leukaemia, brain, and pancreas cancer cells. Our study suggests that 3D-NAF-1^44-67-6K could be used, alone or in drug combinations, to treat ovarian cancer and improve patient survival.

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerative diseases, including Alzheimer Disease. The molecular processes underlying aggregation in disease are poorly understood. Here, we introduce FibrilPaint1 as a tool to measure the size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils. FibrilPaint1 is a 22mer peptide with many exciting properties, which makes it a tool for diagnostics and an attractive start point for developing a class of effective fibril targeting degraders: (i) FibrilPaint1 binds fibrils with nanomolar affinity; (ii) it does also bind to oligomeric precursors, down to a size of only 4 layers; (iii) it does not bind to monomers (KD > 100 microM); (iv) it is fluorescently labelled, which allows monitoring and localising interactions. (v) FibrilPaint1 recognises various Tau fibrils, including patient derived fibrils from Alzheimer, Corticobasal degeneration and Frontotemporal dementia; (vi) FibrilPaint1 is selective for the amyloid state and does not have background binding to amorphous aggregates, blood serum or cell lysate. In combination with Flow Induced Dispersion Analysis (FIDA), a microfluidics technology, we determined the molecular size of amyloid fibrils with sub-microliter sample volumes. This set-up acts as a molecular ruler at layer resolution - we determined Tau fibril length from 4 to 1100 layers in solution. This is an interesting parameter that can be used for diagnostic applications and biochemical research in dementia.

Specific phosphorylation patterns control the activity of multiphosphorylated proteins. In case of the Tau protein, multiphosphorylation leads to the formation of different disease-related condensates and aggregates. Studying the role of these specific patterns at the protein level is crucial for understanding the molecular mechanisms of Tauopathies such as Alzheimers Disease. However, due to the extreme difficulty in obtaining recombinant proteins with specific phosphorylation patterns using kinase-based methods, it is practically impossible to study the connection between specific phosphorylation patterns and aggregation events at the protein level. Here we addressed this problem by reducing the system to the peptide level and studying the effect of specific phosphorylation patterns on the condensation and aggregation of a specific domain of Tau, R4 (residues 336-358). To achieve this aim, we have applied advanced methods to synthesize a library of multiphosphorylated peptides derived from R4. We showed that specific phosphorylation patterns stringently control the formation of Tau aggregates and condensates. Phosphorylation of Ser341 promoted aggregation of R4 while phosphorylation of Ser352 promoted its condensation. Interestingly, Ser356 phosphorylation inhibited both processes, which can be overridden by double-phosphorylation at Ser341/Ser352. Differences between the microenvironments of the phosphorylated residues lead to their different effects on R4 aggregation upon phosphorylation. Our results show that working at the domain level using advanced peptide synthesis methods is a highly useful and practical way to provide valuable information about the effects of post translational modifications on protein activity.

Neurodegenerative diseases are characterised by the formation and accumulation
of protein fibrils. The mechanism underlaying this aggregation process remains
poorly understood. Fibril fragmentation, resulting in seed generation, plays a role in
toxicity. Here we provide a quantitative picture of the impact of ultrasound on
patient-derived and recombinant fibrils from various diseases. Fragmentation of
recombinant Tau fibrils and patient-derived fibrils from Alzheimer’s Disease,
Corticobasal Degeneration and Frontotemporal Dementia generates amyloid and
non-amyloid species. Interestingly, patient-derived fibrils are more susceptible to
ultrasound than artificial fibrils. Understanding fibril fragmentation and the
generation and nature of seeds may provide insights to the molecular mechanism
of the disease progression, contributing to the development therapeutic
approaches.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20–30 % flexible, 30–40 % aliphatic and 20–30 % aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation.

Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association and protein–protein interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered domain (STIL_400–700) and a structured CCD (STIL CCD_718–749). Disrupting STIL oligomerization via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (K_D = 8 ± 2 µM) and tetramerization (K_D = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order oligomers result in increased thermal stability and cooperativity of association. We suggest that this complex oligomerization mechanism regulates the activated levels of STIL in the cell and during centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.

Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-1^44-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-1^44–67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-1^44–67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-1^44–67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-1^44–67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-1^44–67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-1^44–67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

One of the most important properties of intrinsically disordered proteins is their ability to undergo liquid-liquid phase separation and form droplets. The Adenomatous Polyposis Coli (APC) protein is an IDP that plays a key role in Wnt signaling and mutations in Apc initiate cancer. APC forms droplets via its 20R domains and self-association domain (ASAD) and in the context of Axin. However, the mechanism involved is unknown. Here, we used peptides to study the molecular mechanism and regulation of APC droplet formation. We found that a peptide derived from the ASAD of APC-formed droplets. Peptide array screening showed that the ASAD bound other APC peptides corresponding to the 20R3 and 20R5 domains. We discovered that the 20R3/5 peptides also formed droplets by themselves and mapped specific residues within 20R3/5 that are necessary for droplet formation. When incubated together, the ASAD and 20R3/5 did not form droplets. Thus, the interaction of the ASAD with 20R3 and 20R5 may regulate the droplet formation as a means of regulating different cellular functions. Phosphorylation of 20R3 or 20R5 at specific residues prevented droplet formation of 20R3/5. Our results reveal that phosphorylation and the ability to undergo liquid-liquid phase separation, which are both important properties of intrinsically disordered proteins, are related to each other in APC. Phosphorylation inhibited the liquid-liquid phase separation of APC, acting as an ‘on-off’ switch for droplet formation. Phosphorylation may thus be a common mechanism regulating LLPS in intrinsically disordered proteins.

Abnormal kinase activity is highly associated with disease, especially cancer. Thus, kinases are important targets for developing anti-cancer drugs. The common approach for kinase inhibition is using small molecules that compete with ATP for binding the ATP-binding site of the kinase. However, since the ATP-binding site is in many cases common to numerous kinases, it is difficult to achieve selectivity when targeting this site. Here we present an alternative approach of targeting the protein-protein interactions of kinases as means for achieving selectivity in their inhibition. We demonstrate this approach by using peptides for inhibiting the docking D-recruitment site (DRS) of the kinase ERK2. We designed a library of peptides, derived from DRS binding sequences of ERK2-binding proteins. We synthesized the peptides and quantified their interactions with ERK2. Three peptides, derived from the proteins ELK1, SAP1 and SAP2, bound ERK2 in the low micromolar range. These peptides also inhibited the interaction of ERK2 with a surface-bound ELK1 derived peptide. The peptides penetrated HT29 colon cancer cells and induced a moderate decrease in cell viability. Our approach can be further utilized for developing selective peptide-based kinase inhibitors.

Protein aggregation correlates with many human diseases. Protein aggregates differ in shape, ranging from amorphous aggregates to amyloid fibrils. Possibly for such heterogeneity, strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we present a new strategy by which we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. Thus, they act on dynamic precursors before a mechanistic differentiation takes place. Using a peptide array approach, we first identified peptides inhibiting the predominantly amorphous aggregation of a molten globular, aggregation-prone protein, a thermolabile mutant of the Axin tumor suppressor. A series of optimization steps revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants. The key properties that made a peptide active were a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1 and an even distribution of residues of different nature throughout the sequence. Remarkably, the optimized peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer’s disease, and entirely unrelated to Axin. Our compounds thus target early aggregation stages, independent of the aggregation mechanism, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be attractive lead compounds against multiple protein aggregation diseases.

Kinases are responsible for regulating cellular and physiological processes, and abnormal kinase activity is associated with various diseases. Therefore, kinases are being used as biomarkers for disease and developing methods for their sensing is highly important. Usually more than one kinase is involved in phosphorylating a target protein. However, kinase detection methods usually detect the activity of only one specific kinase. Here we describe an electrochemical kinase sensing tool for the selective detection of two kinases using the same target peptide. We demonstrate the sensing of kinases ERK2 and PKCδ. This is based on a single sensing element, a peptide that contains two distinct phosphorylation sites of these two kinases. Reversibility experiments with alkaline phosphatase and reaction with the electrochemically active ferrocene-labeled ATP showed that the mechanism of sensing is by detecting the enzymatic phosphorylation. Our approach can be further utilized to develop devices for the detection of multiple kinases and can be expanded to other types of enzymes involved in disease.

Preparing phosphorylated peptides with multiple adjacent phosphorylations is synthetically difficult, leads to β-elimination, results in low yields, and is extremely slow. We combined synthetic chemical methodologies with computational studies and engineering approaches to develop a strategy that takes advantage of fast stirring, high temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to produce multiphosphorylated peptides at an extremely rapid time and high purity.