Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations. In order to advance our understanding of the regulation of Ubiquitin-specific protease 8 (USP8), which is known to play a role in Cushing’s Disease, Kakihara et al identify and characterise amino acids 645–684 of USP8, which serve as an autoinhibitory region. Their pull-down and single-molecule FRET analysis, as well as in silico modelling, suggest that the release of USP8 autoinhibition may underlie Cushing’s disease. USP8 also maintains the protein levels and/or functions of endocytosis machinery components, including Eps15, HRS, STAM1/2 and CHMP proteins12. In addition, USP8 regulates autophagy and mitophagy by deubiquitinating EPG5, p62 and Parkin15. It suppresses the endoplasmic reticulum export of procollagen by deubiquitinating COPII protein Sec3116 and enables cytokinesis, probably by deubiquitinating VAMP8—a SNARE required for vesicle fusion in cytokinesis17. Given the several functions of USP8, the regulatory mechanism of its enzyme activity must be elucidated; however, limited mechanistic data have been reported till date.Ubiquitin-specific protease 8 (USP8), a member of the USP family of deubiquitinases, regulates multiple membrane trafficking pathways and membrane fusion/fission events. It deubiquitinates endosomal membrane proteins, including epidermal growth factor receptor (EGFR), which enhances their recycling to the plasma membrane and/or inhibits their lysosomal degradation19 , which are utilised for ubiquitin recognition in other USPs, are positioned in a closed conformation. Furthermore, the fingers subdomain is tightened inwardly, making the ubiquitin-binding pocket too narrow to capture ubiquitin. Despite these unfavourable features, the USP domain effectively catalyses the deubiquitination reaction20, implying the occurrence of substrate-induced conformational changes.Similar to other USPs, USP8 harbours a catalytic USP domain comprising three subdomains, the fingers, palm and thumb19 Fig. . These s25 or other domains28 of USPs. The binding of USP8, which has a phosphorylation-dependent 14-3-3-binding motif 30. In the mitotic M-phase, when USP8 positively regulates cytokinesis, it dissociates from 14-3-3 and shows increased enzyme activity29. On the other hand, in corticotroph adenomas of Cushing’s disease, ~50% of tumour samples show somatic mutations of USP8 that cause deletion or substitution of amino acids around the 14-3-3-binding motif32. Cushing’s disease begins with the hypersecretion of adrenocorticotropic hormone (ACTH) from corticotrophs, followed by excess adrenal glucocorticoid. In cultured corticotrophs, the exogenous expression of USP8 lacking functional 14-3-3-binding motifs effectively enhances ACTH secretion31, suggesting that 14-3-3-binding motif dysfunction causes this disease. Thus, 14-3-3 plays important role in regulating USP8 under physiological and pathological conditions. However, the molecular basis of 14-3-3-dependent inhibition of USP8’s enzyme activity remains unclear.Some USPs are activated by specific interacting proteins that bind to the catalytictif Fig. , to 14-3This study aimed to elucidate the regulatory mechanism of USP8 enzyme activity. We identified a WW-like domain in USP8 as an autoinhibitory region. This WW-like domain and ubiquitin were found to competitively bind to the ubiquitin-binding pocket of USP8. In addition, 14-3-3 inhibited the USP8 enzyme activity partly by enhancing the interaction between the WW-like and catalytic domains.Δ414–714) and USP8Δ605–714  and other mutants, suggesting that aa 605–714 contains one or more autoinhibitory regions that decrease the deubiquitinating activity of USP8. Further mutational analyses determined aa 645–684 be a putative autoinhibitory region , which can be irreversibly conjugated to the Cys residue of the catalytic triad33. Ub-VME was able to label USP8Δ645–684 more effectively than it labelled USP8WT , meaning that it shares a common feature with atypical WW domains.The amino acid sequence of the autoinhibitory region is well conserved among vertebrates Fig.  and show35 as a template. In this model, the overall structure of the autoinhibitory region of USP8 is similar to that of canonical WW domains: a three-stranded antiparallel β-sheet34   simulations showed the stability of the model structure. The results also revealed that the substitution of Trp655 to Ser (W655S) abolishes one of three β-strands and destabilises the domain structure ure Fig. . Thus, t655 is substituted to Ser (USP8W655S). We found that USP8W655S shows higher activity than USP8WT  with the USP domain, whereas the deletion of the WW-like domain and the substitution of Trp655 to Ser abolished the interaction , EYFP and ECFP. Specifically, EYFP was fused to the N-terminus of the WW-like domain, whereas ECFP was inserted into five different surface loops of the USP domain  assay Fig.  by constain Fig. . The insWe prepared lysates of cells expressing these probes and measured the emission spectra with the excitation wavelength for ECFP. As negative controls, we trypsinised the lysates to separate EYFP and ECFP in the probes, and then we measured their emission spectra. All probes showed significant FRET signals 20, the predicted structure showed an open conformation in which the USP domain grabs the ubiquitin molecule with the tip of the fingers and the catalytic cleft  eft Fig. . Lys913 18) Fig.  also mak1014 in the BL1 to Ala (F1014A) significantly decreased the FRET signal  by examining the effects of point mutations in FRIP8 on the FRET signal. Results showed that substitution of Phenal Fig. . In addinal Fig. . These f1014 is one of the binding sites and that the inter-domain interaction is the most favourable possibility  decreased the FRIP8 FRET signal, although single amino acid substitutions with each residue separately had no effect  is a common USP8 mutation in Cushing’s disease that causes dissociation from the 14-3-3 protein38. The loss of polar contact between the phosphate group at Ser718 and a basic pocket of 14-3-3 can explain the dissociation30. A previous report indicated that the Ser718 deletion enhances the deubiquitinating activity of USP831. We compared the effects of Ser718 or the WW-like domain deletion on the deubiquitinating activity of USP8. The deletion of Ser718 enhanced this activity more weakly than the deletion of the WW-like domain  in ligand proteins43. Among these, USP25 had previously been the only USP for which the activity was inhibited on a well-defined structural basis. The present study provides another example of the autoinhibition of USPs by an intrinsic region. In USP25, the autoinhibitory region occupies a space between the α5 helix and BL243. Interestingly, the autoinhibitory regions of USP8 and USP25 similarly plug an entrance to the catalytic cleft in which the ubiquitin C-terminal tail should be embedded. In a case where a USP was inhibited by a compound, a similar space in USP14 was occupied by the small molecule inhibitor IU144. Therefore, this space could potentially be utilised for the effective inhibition of various USPs by intrinsic regulatory motifs and chemical inhibitors.Several USPs are reported to be regulated by the intrinsic regions that physically interact with their catalytic domainsWe found that most cellular USP8s are bound to 14-3-3. Our analyses also indicated that 14-3-3 inhibits USP8’s deubiquitinating activity by enhancing the interaction between the WW-like and USP domains. Because the 14-3-3-binding motif is located at an unstructured region between the WW-like and USP domains, one possibility is that binding shortens the distance between these domains and thereby enhances their complex formation Fig. . However31. This study revealed a possible part of the pathogenic mechanism underlying Cushing’s disease: the inability of USP8 to bind to 14-3-3 decreases the interaction between the WW-like and USP domains, which causes USP8 hyperactivation  were used in the two studies, C40 may be produced in a cell type-specific manner. It will, therefore, be important to determine whether significant levels of C40 are produced in the corticotrophs of patients with Cushing’s disease. Nevertheless, our study demonstrated that Cushing’s disease-associated mutations release USP8 autoinhibition, which causes high enzymatic activity even without its cleavage.The 14-3-3-binding motif is a hot spot at which somatic mutations frequently occur in Cushing’s diseaseion Fig. . To dateIn conclusion, we found that the USP8 WW-like domain functions as an autoinhibitory region by binding to the ubiquitin-binding pocket of the catalytic domain. In addition, we found that 14-3-3 binds to USP8 and thus enhances the autoinhibitory interaction. In cells, USP8 deubiquitinating activity might be tuned by the association and dissociation of 14-3-3. In Cushing’s disease, this regulation is apparently impaired by disease-associated mutations at the 14-3-3-binding motif. To build upon our findings, further studies will be required to determine the physiological and pathological significance of this regulatory mechanism of USP8.31, human 14-3-3ε cDNA29, mouse STAM1 cDNA45 and mouse STAM2 cDNA46 were prepared elsewhere. Human Parkin and ubiquitin cDNAs were kindly donated by Drs Koji Yamano and Toshiaki Suzuki, respectively . For the construction of expression plasmids, these cDNAs were subcloned into the following vectors: pME31 for the expression of C-terminal FLAG-tagged USP8; pCDH-CMV-MCS-EF1-Puro (System Biosciences) for the viral infection and expression of N-terminal FLAG-tagged USP8; pcDNA3 (Invitrogen) for N-terminal HA-tagged ubiquitin; pFLAG-CMV2 (Sigma Aldrich) for N-terminal FLAG-tagged USP8, Parkin, STAM1 and STAM2; pmyc-CMV5  for N-terminal myc-tagged USP8 and 14-3-3ε and pGEX-6P2  for the bacterial expression of N-terminal GST-tagged USP domain (aa 756–1118).Human USP8 cDNA48. For ECFP insertion, the restriction sites for EcoRI and BamHI were inserted in the following locations of the USP domain: gaps between aa 816 and 817 as insert-1, aa 898 and 899 as insert-2 (FRIP8), aa 959 and 960 as insert-3, aa 1045 and 1046 as insert-4 and aa 1074 and 1075 as insert-519. ECFP with the spacers was subcloned in a stepwise manner into these EcoRI and BamHI sites. For the construction of His-tagged FRIP8, His-tag sequence was fused to the C-terminal of FRIP8. Site-specific mutagenesis was achieved using PCR with a Prime STAR Mutagenesis Basal Kit (Takara).For the construction of single-molecule FRET probes, human USP8 (aa 645–684) was subcloned into pEYFP-N1 (Clonetech). ECFP with flexible spacers comprising a ten-aa peptide (-Gly-Gly-Ser-Ala-Gly-Gly-Ser-Ala-Gly-Gly-) was amplified through PCR using pECFP-N1 (Clonetech)Escherichia coli Rosetta. Protein expression was induced by the stimulation of cells with 0.5 mM IPTG, which was followed by overnight incubation at 15 °C. Cells were then lysed in ice-cold PBS supplemented with 1% Triton X-100 and protease inhibitors . After centrifugation, the supernatants were mixed with glutathione sepharose 4B . After 1-h incubation at 4 °C, beads were washed five times with PBS and then incubated in ice-cold elution buffer A  and 10 mM reduced glutathione; Wako) at 4 °C for 30 min. The purity and concentration of the eluates were validated using SDS-PAGE and CBB staining using Quick CBB (Wako).A GST-tagged USP domain (aa 756–1118) was constructed in His-tagged FRIP8 was expressed in HEK293T cells. Cell culture and transfection are described below. Cells were lysed in ice-cold lysis buffer  supplemented with 10 mM imidazole and protease inhibitors. After centrifugation, the supernatants were mixed with Ni-NTA agarose beads (Qiagen). After 2-h incubation at 4 °C, the beads were washed five times with Tris-buffered saline  and then incubated in ice-cold elution buffer B  at 4 °C for 30 min. The purity was validated using immunoblotting.For the in vitro deubiquitination assay and ubiquitin-VME labelling, FLAG-tagged USP8 (wild-type and mutants) were expressed in HEK293 cells (for ubiquitin-AMC assay) or HEK293T cells (for ubiquitin chain cleavage assay and ubiquitin-VME labelling). Cell lysis and immunoprecipitation were performed as described below. USP8 levels in each sample were compared using immunoblotting, with an equal number of USP8 molecules being used in the assay.2. To stimulate cells with EGF, they were cultured in the presence of 0.5% FBS for 24 h and then incubated with 100 ng/ml human EGF (PeproTech) at 37 °C for 5 min.HEK293, HEK293T and HeLa cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum (FBS), 100 units/ml of penicillin and 0.1 mg/ml of streptomycin at 37 °C and 5% COPlasmid transfections were performed using polyethyleneimine (Polyscience) according to the standard protocol. Cells were subjected to analyses 24–48 h after transfection.For lentivirus infection, HEK293T cells were transfected with pCDH-CMV-FLAG-USP8-EF1-Puro, psPAX2 (Addgene #12260) and pCMV-VSV-G (Addgene #8454). After 2 days from the transfection, the medium containing the virus was collected and filtrated. HeLa cells were infected with the virus medium in the presence of 8 μg/ml polybrene . After 2 days from the infection, the cells were cultured in the presence of 0.8 µg/ml puromycin. Surviving cells were used for experiments.N-ethylmaleimide (Sigma Aldrich) was added. For the immunoprecipitation of FLAG-tagged USP8 for the deubiquitination assay, 1 mM DTT  was added. After the centrifugation of the lysates, the supernatants were collected. To examine the effects of R18 peptide (Sigma Aldrich) on the interaction of 14-3-3 and USP8 or FRIP8, the lysates were incubated with 100 μM R18 peptide at 4 °C for 30 min.Cells were lysed with ice-cold lysis buffer  supplemented with protein inhibitors. To detect ubiquitin signals by immunoblotting, 2 mM Immunoprecipitation was performed following the manufacturer’s recommended procedures. Anti-FLAG M2 antibody-conjugated agarose beads (Sigma Aldrich), anti-EGFR antibody , anti-GFP antibody  and Protein A Sepharose  were used for immunoprecipitation. After immunoprecipitation, the beads were washed five times with lysis buffer. To elute FLAG-tagged proteins, the beads were incubated in TBS with 200 ng/μl FLAG peptide (Sigma Aldrich) at 4 °C for 30 min.Samples were incubated in SDS-PAGE sample buffer  at 98 °C for 5 min. To detect the ubiquitin signals, samples were incubated in NuPAGE LDS sample buffer with a reducing agent (Thermo Scientific) at 37 °C for 20 min. Samples were separated using SDS-PAGE or NuPAGE (Thermo Scientific).49, anti-HRS (1:100)50, anti-STAM1 (1:200)51, anti-α-tubulin , anti-HA , anti-Myc , anti-GFP  and anti-14-3-3  antibodies. The secondary antibodies used were as follows: peroxidase-conjugated anti-mouse, anti-rat and anti-rabbit IgG antibodies . Blots were detected using ECL Prime Western Blotting Detection Reagents  and ImageQuant LAS 4000 Mini .Immunoblotting was performed following standard procedures. The primary antibodies used for immunoblotting were as follows: anti-FLAG , anti-ubiquitin , anti-EGFR , anti-USP8 (1:200)For ubiquitin chain cleavage assay, USP8 (wild-type or the mutants) was incubated with 25 ng/μl Lys63-linked ubiquitin tetramers (Boston Biochem) in TBS with 10 mM DTT at 37 °C for the indicated times in the presence or absence of R18 peptide. Samples were subjected to SDS-PAGE or NuPAGE and then to gel staining with a Silver Stain MS Kit (Wako).For ubiquitin-AMC assay, USP8 was mixed with 1 μM ubiquitin-AMC (Boston Biochem or Life Sensors) and 10 mM DTT in TBS in 96-well medium-binding, flat-bottom, black plates (Thermo Scientific). To examine the effects of R18 peptide, USP8 was incubated with 100 μM R18 peptide at 4 °C for 30 min before the assay. Plates were set on Varioskan LUX (Thermo Scientific) and incubated at 37 °C. During the incubation, the fluorescence intensity of AMC released from ubiquitin was measured at 10-s intervals using 345 and 445 nm as the excitation and emission wavelengths, respectively. The fluorescence intensity of the sample without USP8 was measured as the background and then subtracted from each value. Non-linear regression curves were plotted using the equation for one-phase association using Prism 9 (GraphPad Software).USP8 immunoprecipitates or His-tagged FRIP8 precipitates were incubated with 1 μM ubiquitin-VME (Boston Biochem) in TBS supplemented with 1 mM DTT at 37 °C for 5 or 30 min, respectively. Samples were then subjected to immunoblotting and a FRET assay.HeLa cells on coverslips were fixed with PBS containing 4% paraformaldehyde, permeabilised with PBS containing 0.2% Triton X-100 and blocked with PBS containing 5% FBS. Cells were stained with mouse anti-FLAG antibody  and Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (Invitrogen) based on standard procedures. Coverslips were mounted on slides with Fluoroshield mounting medium (ImmunoBioScience). Fluorescence images were captured with a laser-scanning confocal microscope .https://blast.ncbi.nlm.nih.gov/) was used to identify protein sequences similar to USP8 aa 645–684. Sequence similarity was analysed using ClustalW ; the colours in the alignment followed the ClustalX scheme. Amino acid sequences of human MAGI1 (accession code: Q96QZ7), human SAV1 (Q9H4B6), human WWOX (Q9NZC7) and vertebrate USP8s were based on annotated information present in the UniProt database (https://www.uniprot.org/).Protein BLAST (https://cluspro.org/). The MD simulations of the derived modelled structures were also performed to examine their stabilities in the physiological condition; the details are described below.In silico structural modelling of the WW-like and USP domains and their complex was performed. In brief, (1) homology modelling of the WW-like domain was first performed using the MAGI1 WW domain (PDB ID: 2YSE), (2) the structural model of the USP domain in the ubiquitin-bound form was then constructed using the ubiquitin-bound USP2 USP domain (PDB ID: 2HD5) as the template for homology modelling and (3) the protein–protein docking simulations of the two domains were executed using ClusPro  conditions of T = 300 K and P = 1 atm, using the Berendsen thermostat and barostat58, with a relaxation time of 1 ps, and using the particle mesh Ewald method59 for electrostatic interactions. The simulation length was 1 μs, together with a 2-fs time step using constraining bonds involving hydrogen atoms via the SHAKE algorithm60. The simulation of the W655S mutant was also performed as described above, after replacing the wild-type W655 with Ser using MODELLER52.Using data on the sequence similarity with the MAGI1 WW domain Fig.  and struA structural model of the USP domain (aa 756–1110) was also constructed to investigate potential interactions with the WW-like domain. Because the open form with bound ubiquitin was not solved for USP8, the ubiquitin-bound USP2 USP domain (PDB ID: 2HD5) was used as a template for homology modelling. The modelled structure of the USP8 USP domain with a 76-residue ubiquitin bound to it was then solvated in a simulation box with the dimensions 103 Å × 84 Å × 91 Å  before being simulated for 1 μs, as described above. The stability of the modelled ubiquitin-USP domain complex was observed by the small root mean square distribution on the Cα atom resolution (Cα-RMSD) of the overall structure being < 2.6 Å from that of the initial simulation model. The structure of the USP domain in the last simulation trajectory time step was then used for the subsequent simulation of docking with the WW-like domain. The MD simulation of the apo USP domain in the closed-form was also performed using the crystal structure (PDB ID: 2GFO) as the initial model.61 to generate putative interaction complexes. We chose six model candidates from these complexes using the criterion that the F1014 of the USP domain existed in the interface with the WW-like domain. We then performed a 100-ns MD simulation for each model candidate, as described above. Using the associated MD trajectories, the number of atom contacts, Nc, was calculated as the number of non-hydrogen atom pairs with r < 4 Å, and the binding free energy between the USP and WW-like domains, ΔGbind, was evaluated using the MM-GB/SA module of AMBER 1657. The structure with the largest Nc and the lowest ΔGbind was selected as the most probable complex model  at 5- or 0.5-nm wavelength intervals, respectively, with 433 nm as the excitation wavelength and 460–570 nm as the emission wavelength range. Negative control samples were incubated with 10 ng/μl trypsin (Thermo Scientific) at 4 °C for 60 min, and then fluorescence intensities were measured. To examine the effects of R18 peptide, samples were incubated with 100 μM R18 peptide at 4 °C for 30 min before the assay. In blank wells, assay buffer or lysates derived from cells that did not express FRET probes were also prepared. These fluorescence intensities were measured as background signals and subtracted from the measured values. The FRET ratio was calculated based on the peak intensity at the EYFP emission wavelength divided by that at the ECFP emission wavelength.t-tests or one-way factorial ANOVA followed by Tukey–Kramer multiple comparison post hoc tests. P values of <0.05 were considered significant.Data were presented as means ± standard deviations. Data were analysed using two-tailed Student’s Un-cropped scan data of blots and staining are shown in Supplementary Figs. Further information on research design is available in the Peer Review FileSupplementary InformationReporting SummarySupplementary Data 1Description of Additional Supplementary Files


Cerebrospinal fluid is a clear fluid that occupies the ventricular and subarachnoid spaces within and around the brain and spinal cord. Cerebrospinal fluid is a dynamic signaling milieu that transports nutrients, waste materials and neuroactive substances that are crucial for the development, homeostasis and functionality of the central nervous system. The mechanisms that enable cerebrospinal fluid to simultaneously exert these homeostatic/dynamic functions are not fully understood. SCO-spondin is a large glycoprotein secreted since the early stages of development into the cerebrospinal fluid. Its domain architecture resembles a combination of a matricellular protein and the ligand-binding region of LDL receptor family. The matricellular proteins are a group of extracellular proteins with the capacity to interact with different molecules, such as growth factors, cytokines and cellular receptors; enabling the integration of information to modulate various physiological and pathological processes. In the same way, the LDL receptor family interacts with many ligands, including β-amyloid peptide and different growth factors. The domains similarity suggests that SCO-spondin is a matricellular protein enabled to bind, modulate, and transport different cerebrospinal fluid molecules. SCO-spondin can be found soluble or polymerized into a dynamic threadlike structure called the Reissner fiber, which extends from the diencephalon to the caudal tip of the spinal cord. Reissner fiber continuously moves caudally as new SCO-spondin molecules are added at the cephalic end and are disaggregated at the caudal end. This movement, like a conveyor belt, allows the transport of the bound molecules, thereby increasing their lifespan and action radius. The binding of SCO-spondin to some relevant molecules has already been reported; however, in this review we suggest more than 30 possible binding partners, including peptide β-amyloid and several growth factors. This new perspective characterizes SCO-spondin as a regulator of cerebrospinal fluid activity, explaining its high evolutionary conservation, its apparent multifunctionality, and the lethality or severe malformations, such as hydrocephalus and curved body axis, of knockout embryos. Understanding the regulation and identifying binding partners of SCO-spondin are crucial for better comprehension of cerebrospinal fluid physiology. Cerebrospinal fluid (CSF) is a clear fluid that occupies the ventricular and subarachnoid spaces inside and around the brain and spinal cord. CSF plays an essential role in the homeostasis of the central nervous system (CNS) and its composition must be finely tuned to establish a stable internal milieu. It provides buoyancy and protection to the brain and spinal cord; it transports nutrients, neuroactive substances, and even waste substances for clearance over the entire CNS; and it regulates brain volume, neurogenesis, behavior, and sleep/wake cycles –5.The appearance of the CSF is concomitant with neural tube formation, a shared feature in all vertebrates. At this early stage, amniotic fluid gets trapped inside the neural tube and constitutes the earliest embryonic CSF (eCSF) . After tThe neurogenic and proliferative activities of some of these factors have been reported by inhibition in vivo and in vitro Table . These sMatricellular proteins are modular extracellular proteins with the ability to interact with different ligands, including growth factors, cytokines, proteases, and cell receptors . These pThe size of matricellular proteins is diverse, with tenascin being the largest, with a monomeric size of ~ 250 kDa and oligomers of over a million Daltons, whereas CCN-1 only 35–40 kDa. Independently from their size, a common characteristic of matricellular proteins is their modular structure. Some domains are shared among several matricellular proteins, such as the epidermal growth factor (EGF)-like domain, von Willebrand factor type-C domain (vWF-C), thrombospondin type I repeat (TSR), and a carboxyl-terminal cystine knot (CTCK) motif  and its similarity with members of the spondin family, such as TSP, F-spondin, or R-spondin  Fig. 1)1). SCO-sSince its description in 1860 , the RF In the next sections, we will analyze the principal characteristics of SCO-spondin, including place and regulation of its secretion “SCO-spondin is highly conserved in all chordates, characterized by the presence of a notochord and a hollow neural tube, structures that arise concomitant to the appearance of RF inside this cavity –40. SCO-The SCO is an ancient brain gland, located at the midline of the caudal–dorsal diencephalon Figs. , 4A, B. Immunohistochemical analysis of SCO in 25 vertebrate species shows a similar organization in all species studied , althougA general approach to determine the functions of new proteins is to transfer annotations from well-characterized proteins with similar domains, which works even better when there is co-occurrence of several such domains , 68. SCOAmong the SCO-spondin domains, TSR, vWF-C, EGF-like , and CTCK domains are characteristic of matricellular proteins.The TSR domain (IPR000884) is found in several matricellular proteins, such as TSP, R- and F-spondin and all members of the CCN family Fig. . The rolThere are 27 TSR domains in the vertebrate SCO-spondin protein, suggesting an important role in the biological functions of this protein . In thisAs previously stated, the TSR domain in other proteins is a well-established interacting domain for soluble factors, such as FGF-2 and TGF-β, which are also present in CSF Table . FGF-2 iThe vWF-C (IPR001007) domain, also known as a chordin-like cysteine-rich repeat, is present in several matricellular proteins, including TSP and CCN family members, as well as in other extracellular proteins such as vWF, Chordin family members, and the BMP-binding endothelial regulator.One of the most reported functions for this domain is the regulation of TGF-β and BMPs –84. The Because TGF-β1 and 2 and BMP7 are present in the adult CSF, and BMP activity is detected at the embryonic stage (Table The CTCK domain (IPR006207) is a highly conserved three-dimensional folded domain found in several extracellular proteins, including vWF, several mucins, a wide variety of cytokines , hormones , and CCN matricellular proteins –90. The Several interacting partners of the CTCK domain have been identified in other proteins, such as integrins , perlecan, vitronectin, decorin, and cell-surface heparan sulfate proteoglycans (HSPGs). In all these interactions, CTCK acts as an important domain that determines how these proteins control cell adhesion processes –95. AddiCTCK domain is also involved in the dimerization and polymerization of homo and heterodimers with other proteins containing the same domain  and in tCumulatively, these antecedents suggest a possible interaction between the CTCK domain within SCO-spondin and factors present in CSF Table  with theAll members of the LDLr family share a similar ligand-binding region comprising at least seven LDLrA domains in tandem, followed by two EGF-like domains . The samThe LDLrA domain IPR023415) is distinctive of the LDL receptor family, whose members contain at least seven of these domains in tandem  B, ApoE, reelin, ApoJ (clusterin), TSP, F-spondin, carrier proteins for lipophilic vitamins, proteases/inhibitor complexes, and members of the Wnt family Table  102, 10.Table 3SLRP1 also binds to the amyloid-β peptide (Aβ), whose accumulation in the brain is a hallmark of Alzheimer’s disease. This receptor is expressed in the brain capillaries and is able to transport Aβ across the blood–brain barrier in a concentration-dependent manner. It has been proposed that the main directionality of the Aβ from the brain to the plasma is owing to the presence of soluble LRP1 in the blood, which acts a “sink,” sequestering 70–90% of the plasmatic Aβ, diminishing its concentration, and favoring the directional transfer from the brain to the blood , 105.The similarity between SCO-spondin and the ligand-binding region of the LDLr family suggests that SCO-spondin binds the same molecules, some of which are present in CSF in normal or pathological conditions, such as Aβ, lipoproteins, clusterin, or reelin Table . In thisIn addition to the binding capacities, a hypomorphic missense mutation that disrupts evolutionary conserved cysteine at LDL domain  revealedThe EGF-like domain IPR000742) has been linked to several biological functions and is able to bind different extracellular molecules as well as cellular receptors. In addition to the LDLr family, this domain is also present in some matricellular proteins, such as tenascin and TSP. In TSP, the third EGF-like domain is responsible for FGF-2 binding ; in tena has beenSCO-spondin contains two EGF-like domains following the LDLrA domains, resembling the ligand-binding region of the LDL receptor family. Additionally, this SCO-spondin domain may bind soluble FGF-2 in CSF or the EGF receptor expressed on ependymal cells and in subventricular neurogenic niches , where tAs stated above, SCO-spondin can be found soluble in CSF or as aggregates in the form of RF, an elastic threadlike structure. The process of SCO-spondin polymerization has not been elucidated, but it is interesting that the same domains responsible for the polymerization of vWF , a protein capable of forming ultra-long chains of several hundred of monomers, are also present in SCO-spondin. In addition to these domains shared with the vWF, SCO-spondin also contains one EMI domain, also related with polymerization.The cysteine-rich EMI domain typically contains six or seven cysteine residues, which likely form disulfide bonds. The EMI domain has been identified in few proteins, including elastin microfibril interfacer 1 (EMILIN-1) protein, multimerins, NEU1/NG3, periostin, and TGFβ-inducing protein . In all The role of EMI as a multimerization domain critical for RF assembly is also supported by the analysis of a SCO-spondin zebrafish mutant with five extra amino acids in the single EMI domain, which expresses an abnormal protein that fails to form the RF .vWF-D (IPR001846) is a large domain present in few proteins such as otogelin, zonadhesin, different mucins, vWF, and SCO-spondin, all characterized by the generation of multimers by inter and intrachain disulfide bonds.vWF contains four vWF-D domains with a self-organization function Fig. . This prThe TIL domain (IPR002919) is mainly present in trypsin inhibitor proteins; however, it can also be found in other extracellular proteins, including several mucins , the IgGSCO-spondin contains 16 TIL domains, described previously as SCO-spondin repeats , distribIn addition to the protein component, SCO-spondin displays a great variety of different N-glycan structures. Electrophoretic analysis of bovine RF treated with endoglycosidase F shows a decrease (between 10 and 25%) in the molecular mass of its four principal immunoreactive compounds . The preN-glycans to highly complex tetra-antennary N-glycans containing bisected N-acetylglucosamine (GlcNAc), up to three sulfations, and/or several sialic acids of the Neu5Gc or Neu5Ac type . SC. SC28]. The progressive aggregation of SCO-spondin has been detailed in bovine RF. Light and electron microscopy revealed a threadlike structure of 50-µm, composed of bundles of thin filaments of approximately 2–5 µm thickness, which in turn are formed by microfilaments of approximately 10 nm thickness that run longitudinally along the fiber . The thiThe mechanisms that cause this high grade of SCO-spondin polymerization are not fully understood, but it seems to have intra and extracellular components. At intracellular level, pulse–chase assays after intraventricular injection 35S-cysteine in adult rats –156. In At the extracellular level, the progressive polymerization of SCO-spondin is CSF dependent. This requirement seems to have the following three components: first, SCO-spondin may form oligomers at the intracellular level, but the formation of interchain disulfide bridges seems to require other CSF proteins, such as galectin . Second,Once formed, RF exists in a state of continuous movement via the addition of new molecules at its cephalic end, which progressively advance until their disaggregation, several days later, at the caudal region of the spinal cord . In thisIn summary, the formation of the RF is a complex process, which may require initial intracellular SCO-spondin oligomerization, extracellular SCO-spondin cleavage, interaction with galectin, and polymerization in growing structures  dependent on CSF flow.The function of the SCO and its secreted product, SCO-spondin, has remained elusive for more than a century. Multiple possible functions have been attributed, with the most relevant being different aspects related with morphogenesis, CSF cleaning and transport, maintenance of CSF flow, and prevention of hydrocephalus , 31, 38.SCO-spondin is expressed early during development, but at a variable stage depending on the species; for instance, 17 h post-fertilization (hpf) in zebrafish, 3.5 days in chick, and 14 days in rat , 163. InThe inhibition of SCO-spondin using diverse approaches showed the relevance of this protein at various embryonic stages and different animal models Fig. . In thisThe relationship between abnormal RF and curved body axis has been historically reported in fishes and lizards , 168 altTranscriptomic analysis of sspo zebrafish mutants , 148 revSCO-spondin also participates in axon guidance in the posterior commissure (PC). This commissure is located between the basal processes of SCO cells, and immunohistochemical analysis suggested that SCO-spondin is also secreted by these processes toward the extracellular space, aiding in the guidance and fasciculation of the PC axons , 169–171In summary, SCO-spondin is crucial in morphogenesis, and its mutation causes severe malformations and embryonic lethality. These malformations are found in the cephalic cavity of embryos in which SCO-spondin remains soluble (e.g. in chick) and in the spinal cord of embryos where the SCO-spondin rapidly aggregates to form the RF (e.g. in zebrafish). These malformations can be explained, at least partially, by the ability of SCO-spondin to bind LDL , epinephCSF flow depends on multiple factors, such as CSF production by the choroid plexus, ciliary beating of ependymal cells, heartbeat, and pulsatile and local exchange among interstitial fluid, blood, and CSF , 172. InAs stated in the introduction, CSF composition must be finely tuned, establishing a stable internal milieu for the brain, but it is a dynamic fluid, which transports nutrients, neuroactive substances, and waste substances for clearance across the entire CNS. This apparent paradox can be explained by mechanisms capable of binding and releasing CSF factors depending on physiological context. SCO-spondin and the RF appear to be optimally suited for this job owing to the multiple potential binding sites for CSF factors (Table To date, there is evidence regarding the binding of monoamines  and LDL The function of SCO-spondin as matricellular protein is also supported by transcriptome analysis of sspo mutant zebrafish. It is well-established that the loss of a gene in a mutant animal is frequently compensated for by another with overlapping functions, triggering a transcriptional adaptive response . In thisdmh4/+ mutant zebrafish [The relevance of SCO-spondin in CSF homeostasis is also suggested in scoliotic scospondinebrafish . These aebrafish . Having The detoxification role of SCO-spondin has also been suggested in animal models of copper , aluminuThe background set out above suggests that SCO-spondin is a giant matricellular protein capable of maintaining homeostasis in CSF by modulating and trapping several binding factors and permitting the physical dynamism necessary for transporting and releasing the bound molecules in different spatial, temporal, and biological contexts. The binding of SCO-spondin to some relevant molecules has been already reported; however, considering that other smaller matricellular proteins bind more than 80 different molecules, it seems that only the tip of the iceberg of binding partners has been discovered. In this review, we suggest more than 30 possible binding partners (Table In relation to the SCO-spondin in humans the information is contradictory. Human SCO is well developed during fetal and neonatal stages, but it progressively regresses at posterior stages . The secFull understanding of SCO-spondin properties, including its structural conformation, regulation, and behavior in different contexts, will lead to a better comprehension of CSF physiology and open possibilities to new therapeutic tools for treating pathologies. In fact, the binding capacities of matricellular proteins and GAGs have already been exploited in drug delivery for the treatment of different diseases , 189.In summary, SCO-spondin is an incredible protein, highly conserved, highly glycosylated, highly disordered, with several isoforms and enormous size. The study of this protein is complex but extremely relevant. Thus, with this review, we aim to motivate new researchers in the field to better understand this ancient and versatile protein.


Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and—in contrast to neurons—does not impact on glomerular development or maintenance. The atypical cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase with prime functions in terminally differentiated cell types . In neurIn the kidney, Cdk5 is exclusively expressed in glomerular epithelial cells called podocytes . PodocytIn podocytes, Cdk5 is activated by p25, p35, and/or cyclin I, which also determine the subcellular localization of the kinase . DependiTo investigate a potential developmental role of Cdk5, which may be referred by yet unknown activators of the kinase, we generated podocyte-specific Cdk5 knockout mice and analyzed these animals under non-stressed conditions. In addition, we delineated the contribution of Cdk5 in states of glomerular disease, employing inducible podocyte-specific Cdk5 knockout mice and the NTN disease model.Podocyte-specific Cdk5 knockout was generated by mating Cdk5flox mice obtained from Jackson Laboratory  with hNPHS2.Cre mice ,26. TranFor the assessment of Cdk5 knockout efficacy, Cdk5flox mice were mated with mPodocin.2A.Cre.2A.mTomato mice, yielding mTomato-fluorescent podocytes in the offspring . GlomeruIn all animal studies, only male mice were employed. Mice were bred into mixed FVB/N and 129S4/SvJae backgrounds and maintained under standardized, pathogen-free conditions in the University of Washington animal facility, as well as in the University of Cologne animal facility. The experimental protocol was reviewed and approved by the Animal Care Committee of the University of Washington, Seattle, USA and by the State Agency for Nature, Environment, and Consumer Protection, North Rhine-Westphalia, Germany .Temporal control of Cdk5 knockout in podocytes was achieved by mating Cdk5flox mice with hNPHS/rtTA/TetO-Cre mice. In the offspring, transgene transcription was controlled by a podocin-driven promoter which expressed Cre recombinase specifically in podocytes in the presence of doxycycline . DoxycycNephrotoxic nephritis was induced by intraperitoneal injections of sheep anti-rabbit glomerular antibody, as previously described . Fourteepko) and wild type mice and stained with primary antibodies listed in the Immunohistochemical analyses were performed on kidney sections of podocyte-specific Cdk5-knockout (Cdk5ipko), wt; n = 6 each). Podocyte number was quantified on stained paraffin-embedded kidney sections with a specific primary antibody against Wilms’ tumor 1 protein (WT1) . Six anin = 6). In addition, the glomerulosclerosis index was assessed. To this end, 50 glomeruli minimum per individual animal were categorized based on their percentage of scarred glomerular tuft area, divided into 4 groups: grade 1, <25%; grade 2, 25–50%; grade 3, 50–75%; and grade 4, 75–100% [Glomerulosclerosis was quantified on paraffin-embedded kidney sections stained with periodic acid-Schiff reagent as percentage of involved glomeruli per representative section, in all individual groups  and further assessed with ImageScope (Aperio) image-processing software v12.1 .wt/wt fructose (1 mL of dH2O added to 4 g of fructose) at 37 °C for 15 min and then mounted in a MatTek dish with a cover slip on top, prior to imaging with a Leica SP8 3× gSTED system  using a 100× 1.4 NA objective. To quantify the slit diaphragm length per area, a previously published ImageJ macro was used [A simplified version of a previously published protocol was used . Formaldwas used .For the assessment of proteinuria, spot urine was collected from animals of each cohort at day 0, as baseline, and at the following days as indicated. Initial analyses were performed by Coomassie blue staining after SDS-PAGE of small volumes of urine. Quantitative assessment was performed by measuring urinary protein using the sulfosalicylic acid method  and urinStatistical calculations were performed with PalmPrism. For histological assessment, Image Scope  was used.pko) were generated by crossing mice carrying the floxed Cdk5 allele with mice containing Cre recombinase under the podocyte-specific human podocin promoter (pko), heterozygous Cdk5-floxed (Cdk5het), or homozygous wild type Cdk5 (Cdk5wt) alleles. Expression of Cdk5 was reduced to 46.31% (SD 11.86) in Cdk5het and 13.1% (SD 2.26) in Cdk5pko mice, compared to Cdk5 wild type littermates . Phenotyping included screening for proteinuria, and kidney histology was performed in Cdk5pko mice at 70 weeks of age. Coomassie staining of urine samples showed no detectable proteinuria, specifically, no albuminuria  were generated  vs. Cdk5contr: 1.37 (SD 0.16); p < 0.05)  (ipko mice (Cdk5ipko: 62.46 g/g (SD 15.35) vs. Cdk5contr: 22.11 g/g (SD 5.10); p < 0.05). No significant proteinuria was detected in control saline-treated mice  in mice 10 weeks of age A. Histol animals B. Glomer < 0.05) C. In add < 0.05) D. No glo C,D. Nephted mice E.Previously, we and others established the pro-survival signals referred by Cdk5 in podocytes and other post-mitotic cells ,19,20,34ipko mice or controls prior to disease induction (day 0)  staining, was not detected in Cdk5 (day 0) A,B. At das noted C,D. In d control E,F. Evenpko vs. control, reflected by the regular glomerular architecture and function, i.e., absence of proteinuria, argues against a role of Cdk5 in podocyte development.Cdk5 is an ubiquitously expressed proline-directed serine/threonine kinase implicated in both physiological and pathological cellular processes. Amongst these are cytokine production, regulation of insulin levels, migration and invasion, angiogenesis, myogenesis, apoptosis, and senescence (reviewed in Sharma and Sicinski ). ImportRecently, several studies in podocytes investigated the beneficial role of the intermediate filament protein nestin in states of stress and its regulation of Cdk5/p35 inhibition of apoptosis ,44,45. TIn conclusion, our study demonstrated that Cdk5 was not implicated in podocyte development but served a central role in the regulation of podocyte apoptosis. Cdk5 activity is crucial after toxic stimuli to avoid initiation of apoptosis, cytoskeletal remodeling, detachment from the glomerular basement membrane, and, eventually, loss of podocytes in the urine. Several studies on Cdk5 in neurological and oncological disorders showed good transferability of results gained in mice to human disease ,54. Cons


Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far , even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses. Si. Si20]. P domain ,81. HoweL domain . Interesmain . Th. ThGag cP(F/S)AP , the domP(F/S)AP  25]. Mu. MuGag cmultivesicular bodies (MVBs) biogenesis  1 to 7 , the ESCRT-associated protein ALIX, and the AAA ATPase VPS4 , LEM domain 2 protein (LEMD2), and arrestin domain-containing protein 1 (ARRDC1)  and areiew, see . In metaiew, see ,101, areiew, see  and ARRDiew, see , to recriew, see ,106,107.iew, see . Interesiew, see ,110,111 iew, see , as welliew, see ,112,113.iew, see ,114,115,iew, see   budding, and conversely, depletion of ESCRT-I had little effect on ASV release but blocked HIV-1 budding, suggesting that ASV and HIV-1 Gag proteins use different combinations of ESCRT proteins to promote budding [Gag) were also found to interact in vitro and ex vivo via the PTAPP motif in the p6 domain of the precursor .nL L domain, and in this case, the overexpression of the V domain of ALIX did not affect viral particle release [nL motif in patients in therapy failure [simian immunodeficiency virus (SIV) (such as the SIV rhesus macaque and the SIV African green monkeys) lack YPXnL motif, although they can bind ALIX. Indeed, crystal structures revealed that in this case anchoring Tyr and nearby hydrophobic residues lead to the interaction between the precursors and the V domain of ALIX, thus revealing how lentiviruses employ diverse sequences to bind ALIX to efficiently promote virus budding [As mentioned above, the HIV-1 subtype C (HIV-1C) naturally lacks the YPX release . In this failure  was foun failure ,85. Simi budding .herpes simplex virus type 1 (HSV-1). Indeed, these findings suggested the presence of alternative mechanisms to recruit the ESCRT-III proteins that provide the scission of the membrane and thus the release viral particles [Finally, the suppression of ALIX and TSG101 mediated by RNA interference (RNAi) was also enabled to clarify the contribution of these factors in the production of a DNA virus, the articles .Gag precursor interacts with the WW domains of several members of NEDD4 family  . The re budding . Similar budding . Further budding . Interes defects , thus deRetroviridae, as for instance rhabdoviruses and filoviruses [Gag does not contain a PPxY L domain, the overexpression of catalytically active NEDD4-2 (NEDD4L) E3 ubiquitin ligase can rescue the budding of an HIV-1 construct where both PTAP and YPXnL L motifs were deleted [angiomotin (AMOT) [arrestin-related trafficking (ART) proteins [The interaction between PPxY and the WW domain in HECT E3 ubiquitin ligases also regulates the budding of several viruses outside the oviruses . These voviruses ,165,166.oviruses . Interes deleted ,168. Furn (AMOT)  , poliovirus, rotavirus, and norovirus [enveloped HAV virions (eHAV) also depends on the ESCRT components ALIX, VPS4, CHMP2, and IST1 [It was recently shown that several non-enveloped viruses such as orovirus ,6,7 can orovirus ). Intereand IST1 ,173,174.and IST1 ,172. Mutand IST1 . Interesand IST1 , and pX and IST1  and viraGag precursor [Mayaro virus (MAYV), and HSV by covalently binding the UEV domain of TSG101, which results in the disruption of TSG101–ubiquitin interaction [Enveloped RNA viruses derive their envelope from the host as they bud through cellular membranes, and viral budding requires the deformation of the cellular membrane followed by a fission event allowing the generation of distinct cellular and viral membranes. Many viruses therefore hijack the cellular ESCRT machinery to remodel the membranes at viral budding sites, thereby enabling the release of infectious enveloped viral particles. To recruit the ESCRT factors, enveloped viruses use conserved viral assembly L domains encoded by their multifunctional structural proteins. Three types of L domains have been characterized thus far: P(T/S)AP, YPXnL, and PPxY, and two other types, FPIV and PLPPV, have been reported in the M protein of paramyxoviruses ,77,78 anrecursor , respectrecursor ,173,174.recursor ,176. Simeraction ,178,179.


Von Willebrand Factor (vWf) is a giant multimeric extracellular blood plasma involved in hemostasis. In this work we present multi-scale simulations of its three-domains fragment A1A2A3. These three domains are essential for the functional regulation of vWf. Namely the A2 domain hosts the site where the protease ADAMTS13 cleavages the multimeric vWf allowing for its length control that prevents thrombotic conditions. The exposure of the cleavage site follows the elongation/unfolding of the domain that is caused by an increased shear stress in blood. By deploying Lattice Boltzmann molecular dynamics simulations based on the OPEP coarse-grained model for proteins, we investigated at molecular level the unfolding of the A2 domain under the action of a perturbing shear flow. We described the structural steps of this unfolding that mainly concerns the  Von Willebrand Factor (vWf) is a giant extracellular blood plasma protein that plays a key role in arterial hemostasis and thrombosis. Normally, in vivo vWf is a linear multimer consisting of 40–200 covalently linked monomers . With a The three neighbouring globular domains A1, A2 and A3 attract the most attention as they provide important properties related to platelet adhesion and aggregation. A3 domain is responsible for binding to collagen and thus initiation of thrombosis. A1 domain can establish adhesive bonds with GPIb platelet membrane receptors and is crucial for platelet plug formation under high shear stress ,17. MechDuring primary hemostasis in arteries and arterioles, platelet adhesion depends on the length distribution of the vWf multimers ,19,20. TMechanical stability of A2 domain is thus one of the major points of interest with respect to vWf thrombogenic activity and regulation. Characterization of the mechanical properties of vWf at the molecular scale is important for understanding its hemostatic functions. Prior studies underline the resistive role of the central e regime . Structue regime . Howevere regime . Severale regime ,32,33,34e regime ,35,36) ae regime ,38, Optie regime ,33) weree regime ,39,40, ae regime .Further insights emphasize the importance of the structure of globular domains and flexible linker segments for understanding biogenesis, shear-induced conformational changes, platelet recruitment abilities, and mechano-chemical regulation of von Willebrand factor ,41,42. TIn the present work we use computer simulations to study molecular details of the A2 unfolding and the exposure of the scissile bond Tyr1605-Met1606  collisional operator  with a lFor the dynamics of the A1A2A3 system we used a multiple time stepping to separate the integration of the bonded interactions involved in the flexible A2 domain, the A1/A3 elastic network domains and Lde MUPHY . Technicde MUPHY ,58,61. TAll-atom simulations of clamp-force unfolding of the A2 domain were carried out in Gromacs 2018.4 patched with PLUMED 2.5, using the Amber a99SB-disp force-field for disordered and ordered proteins together with its TIP4P-disp water model . The strk:The unfolding kinetics of a protein subjected to a pulling tensile force has been often described using a two-state irreversible Markovian model for the underlying evolution. In this model, the first passage time According to Bell’s model , when an systems . Moreoveordinate . In thatIn order to characterize the unfolding dynamics of the A2 domain under the action of an external pulling force, or fluid shear, we followed the time evolution of several independent runs and introduced a suitable order parameter to describe the time-dependent unfolding. At a given threshold of the order parameter, an unfolding event is recorded, yielding for that simulation a first unfolding time The best estimate n forces , t and To support that intuition we analyzed the number of native hydrogen bonds formed between each The number of contacts for the three pairs of This sequence of unfolding detected in our simulations is consistent with that described in previous work using SMD simulations ,36.It is worth noticing that the cleavage of vWf-A2 is a multistage process that obeys the known “key-lock” principle. The active domain of metalloproteinase ADAMTS13 has relatively low proteolytic activity against human von Willebrand factor, and they are sometimes considered as a non-optimal enzyme-substrate pair . Before recognition site. In case of force-pulling since the force is applied at the C-terminus the recognition site is exposed before the cleavage site, see In the disintegrin-like domain of ADAMTS13 a disordered region called V-loop is present. Although being variable in length and sequence, such region is also found in different ADAM-family proteins . In ADAM2 domain ,70,71. R2 domain . This hy2 domain . Given tIn the previous section we have shown that the OPEP model describes the force induced unfolding paths and kinetics of the isolated A2 domain very similarly to what obtained by an all-atom model in explicit solvent. Here, we investigate a more realistic system, with the A2 domain linked to the neighbouring domains, A1 and A3, as found in the vWf. The complete system is described in the Methods section. In order to study the effect of shear flow we first generated a set of independent initial states by equilibrating the system in the absence of shear perturbation and by extracting a configuration every 1 ns. In these initial configurations the A1 and A3 domains are not in contact with A2, but the linkers are not fully extended. Each configuration was used to start a simulation where the fluid shear flow was activated. First we considered the high shear regime that induces unfolding and exposure of the cleavage site on a timescale of tenths of nanoseconds.The exposure dynamics of The elongation dynamics in shear flow can also be followed by monitoring the chain extension of the A2 domain expressed by its end-to-end distance, see 9 s−1 and a directional mechanical force of 500–700 pN.By using the same strategy adopted for the force-induced unfolding, we quantified the unfolding kinetics by applying the diffusive model (the most adequate to fit the data), see We have then verified the simultaneous exposure of the cleavage and recognition sites, see In this section we relate more explicitly the fluid shear flow to the mechanical tensile force that triggers the unfolding of the A2 domain. We recall that, at longer length scales, and considering specifically the multimeric vWf, the analysis of the tension profile recorded along the vWf chain modelled as a polymer moving under the action of shear flow and hydrodynamics interactions, suggested a general mechanism  that leads to the transition from the collapsed to the elongated shape . By comb ∼109s−1 ,61. At timescale .We now quantify at the level of the A2 terminus, where the unfolding starts, how the action of the shear translates in a mechanical pulling force. In our model the terminal residues of the A2 domain are attached to the initial beads of the linkers LIn nal flow . The actIn 5–106 s−1).In order to better characterize the competitive effects on the three domains of the extensional and rotational components of the shear flow, we built a second model of the A1A2A3 system. In this model, all three domains are described by an elastic network so that also the A2 domain maintains its folded structure. The model allows us to span longer timescales in the simulation since we can increase the integration time-step and reduce the intramolecular degrees of freedom for A2. Therefore we can investigate the overall rotational dynamics of the molecular construct, and the inter-domains interactions, even at small shear rate values  First, we quantify the rotational dynamics of the two peripheral domains, A1 and A3, with respect to the central A2 domain. This is done by computing for each simulated system The obtained TCFs decay in time but their trend do not fit to standard decay models . Therefore, from each TCF, we have individuated the value when it reaches zero the first time. These characteristic times averaged over independent trajectories and as a function of the shear rates are plotted in panel A and B of The rotational dynamics implies that the molecular three-domain system is exposed cyclically, at least at the highest shear rates, to the tensile force and to angular acceleration. The angular velocity of the peripheral domains A1 and A3 with respect to the central A2 is computed by considering the variation of the vector connecting the centers of mass in the unit of time In the same spirit of previous work , but at  scaling ,73). How scaling , and theIn the present paper we used both full-atomistic and coarse-grained computer simulations to explore the unfolding of the A2 domain of vWf in two perturbative regimes typical of experimental setups: the AFM-like unidirectional pulling with a constant force, and the Couette flow typical for near-wall regimes of micro-fluidic flow chambers and rotational viscosimeters. We considered the isolated A2 domain and the A1A2A3 tri-domain construct of the vWf monomer. Our results show that the unfolding pathway of the A2 domain is controlled by the sequential disruption of the The unfolding kinetics under extremely large forces, according to the presented analysis, deviate from Bell’s law and are better approximated by the inverse Gaussian model. The exposure of the Tyr1605-Met1606 cleavage site may correspond to partial unfolding of the A2 domain. However, under stronger forcing, complete unfolding events leading to a fully linear unfolded protein structure have been observed in simulations over tens of nanoseconds timescale.The unfolding pathway of the A2 domain depends on the deformation mode, applied forcing, and, importantly, on whether the individual A2 domain or the tri-domain A1A2A3 structure is loaded. For the A1A2A3 assembly in Couette flow the deformation pathway consists of the stretching of the A2 domain punctuated by relatively long periods of rotation-induced refolding, at least under high shear stress. Due to the tumbling motion, A2 can not only adopt a linear, extended conformation, but also a melted globule one that hinders ADAMTS13 binding and subsequent proteolysis. This contrasts with the behaviour of the individual A2 domain under hydrodynamic shear, in which we mostly detected transient periods of partial unfolding. Moreover, the results of CG simulations suggest that, if the shear stress is strong enough, the rotational component of the flow dominates over the protein’s Brownian motion above a critical threshold. In the shear flow we observed two different scenarios of A2 extension, revealing a complex interplay between fluid dynamics and molecular structure.Several intriguing questions emerge, including the refolding pathway of the A2 domain after deformation, the reversibility of the unfolding, the structure of the compacted globule and influence of the protein’s molecular surroundings on this process. According to , refoldiFinally, by focusing on the relative inter-domain dynamics, we have individuated a shear-rate controlled dynamical configurational transition among a stable collapsed structure of the three domains (low shear) and a cyclically alternated collapsed/elongated structure (high shear). The preferential inter-domain interactions visible in the collapsed structures highlight the key role of these interactions in self-inhibition to cleavage.The present study is a step toward a better understanding of the biophysical mechanisms that govern mechanoreception and mechanotransduction in proteins at the molecular level.


Many types of baked goods are firmly rooted in the food habits of many people in different countries. Although there have been great strides in improving milling, kneading, and baking, given the lack of essential studies, further steps forward need to be taken to understand the effects of storage time and environmental storage conditions, thus motivating this work. The aim of this study is to assess the effects of storage time, using one-way ANOVA, and environmental storage conditions , using MOLS analysis, on flour composition, dough rheology, and biscuit characteristics. Seven levels of storage time were tested: T0 (control), T1, T2, T3, T4, T5, and T6. The results showed that flour storage time significantly increased dough tenacity (P) and curve configuration ratio (P/L), and decreased the biscuit volume (best at T0). However, 2–3 weeks of storage highlighted a significant increase in deformation energy (W), an essential alveograph parameter that is closely correlated to the technological success of leavened products. This optimum found for W might be considered as a great stride in understanding the effects of storage time, confirming that wheat flour can reach its optimal performance after two-three weeks of storage, in particular for W. Moreover, this information could be useful, not only for biscuits production, but also for bread and bakery products . MOLS analysis highlighted that dough rheology and biscuit characteristics are mainly affected by flour composition (primarily from starch content) rather than environmental storage parameters. In conclusion, to optimize the biscuit characteristics, it is necessary to use flours with a low content of damaged starch by selecting the most suitable milling technique and carefully managing the operative parameters. Bread and bakery products are considered worldwide as staple foods, essential for human nutrition. As highlighted in an earlier work , bread, One example might be a traditional Tuscan biscuit named biscotto di Prato. This traditional product takes its name from the municipality of production , a town very close to Florence. However, it is very important not to confuse the biscotto di Prato with the well-known Cantuccini Toscani PGI . In fact, although these biscuits look similar at first glance, they have some important differences. Firstly, Cantuccini Toscani have the PGI denomination, unlike biscotto di Prato. Secondly, these biscuits are produced following different ingredients and recipes , and salt, while biscotto di Prato is produced using only wheat flour, sugar, eggs, almonds and pine nuts). However, despite the different recipes could influence the final products characteristics, as highlighted in earlier works, these products are mainly influenced by the four major stages of the production chain, i.e., milling, kneading, leavening, and baking ,7,8,9,10With respect to the milling process, Cappelli et al. (2020)  summarizAlthough milling has the greatest impact on dough rheology and bread characteristics, the kneading process also plays a key role ,8. CappeSubsequently to kneading, another important unit operation, namely leavening, can significantly affect the bread and bakery products characteristics. As highlighted by Venturi et al. 2021) , the sel , the seAlthough scientific and technological research has made great strides in improving the four major stages of the production chain, given the lack of essential studies, further steps forward need to be taken to understand the effects of storage time and environmental storage conditions, thus motivating this work. In particular, only a few papers regarding the effects of flour storage in controlled conditions for bread production are available in the literature. Moreover, specific papers assessing the effects of flour storage in uncontrolled environment  on flour quality, dough rheology, and biscuit characteristics are lacking, highlighting the novelty of this research. As a result, the aim of this study is to assess the effects of storage time and environmental storage conditions , on flour composition, dough rheological properties, and biscuit characteristics.Triticum aestivum), defined 00 under Italian legislation , white sugar (100% sucrose), almonds, and pine nuts were kindly provided by Biscottificio Antonio Mattei Ltd. . The tested flour, produced from the same batch of wheat, was provided by Molino Cerioli . Immediately after milling, the flour was sent to Biscottificio Antonio Mattei Ltd. to implement the experimental trials. As soon as the flour was received, the first control sample  was used to carry out flour characterization analyses, dough rheological assessment, and biscuit production. Successively, flour was stored in the warehouse of the company using 25-kg paper bags divided into six different pallets (corresponding to the six storage time tested in this work (T1 (one week), T2 (two weeks), T3 (three weeks), T4 (four weeks), T5 (five weeks), and T6 (six weeks))). The aforementioned raw materials were used for the production of the tested biscuits (biscotto di Prato), following the Biscottificio Antonio Mattei traditional recipe and using the machines and plants of the factory located in Prato. A graphical representation of the experiment is reported in Commercial Italian wheat flour  able to detect temperature and humidity variations in the company’s warehouse during the trials. The thermo-hygrometer was placed next to the flour pallets to have a reliable measure of temperature and humidity. Moreover, these parameters were recorded every five hours ), three days a week  for seven weeks (from 27 April 2021 to 12 June 2021).Flours analysis was carried out by the Analytical Food Laboratory , according to AOAC approved methods . In partRheological properties were evaluated using a Chopin NG alveograph, linked to an alveolink integrator–recorder . Consistently with the procedure described in ISO 27971 , dough tThe production process of the tested biscuits (biscotto di Prato), followed the Biscottificio Antonio Mattei traditional recipe using the machines and plants of the factory located in Prato . The biscuits production was carried out every Tuesday, for seven weeks (from 27 April 2021 to 12 June 2021), keeping the same recipe. Moreover, all tests were carried out in three replicates. Following a randomized scheme developed using the statistical software R, from three different bags randomly selected, 7 kg of flour were sampled and placed in a container. Successively, the 21 kg of flour was homogenized, and a representative sample was collected and sent to the Analytical Food laboratory for flour characterization, as reported in Shortly afterwards, the dough divider machine helped to split the mass in several smaller doughs which were subsequently formed using the molder machine. Successively, the small loaves of dough were baked at 230 °C for 18 min. At the end of baking, following a randomized scheme developed using the statistical software R, three baked small loaves were selected to obtain the tested biscuits. In particular, a single biscuit was collected exactly from the center of each small loaf. After one hour cooling, the biscuit volume (L) and height (mm) were measured. The standard millet displacement method . Moreovep < 0.05. In cases of statistically significant results (p < 0.05), a Tukey HSD post hoc test was performed.The effects of storage time were assessed using a one-way ANOVA, which was applied to each alveographic and biscuit parameter to establish its significance. Seven levels were tested: T0 (control), T1 (one week of storage), T2 (two weeks of storage), T3 (three weeks of storage), T4 (four weeks of storage), T5 (five weeks of storage), and T6 (six weeks of storage). Significance was set at 2 of these models, which factor between storage temperature, storage humidity, and flour composition has determined the most significant effects on dough rheology and biscotto di Prato characteristics. The effects of environmental storage conditions, specifically environmental temperature and humidity in the company warehouse, were investigated with a multiple ordinary least square regression (MOLS). MOLS allowed to assess the relationship between rheological properties  and biscuit parameters (volume and height) with flour components  and environmental storage conditions  through the development of specific models. The aim was to determine, according to the RIn particular, the following models were investigated: (1) single alveographic parameters  versus the compositional parameters of the flour ; (2) single alveographic parameters  versus mean temperature and humidity of the week; (3) Biscotto di Prato volume and height versus the compositional parameters of the flour ; (4) Biscotto di Prato volume and height versus mean temperature and humidity of the week. The statistical analysis was carried out using R software.The results of the flours characterization are reported in Flour moisture significantly increases in the first week of storage and then slowly decreases till the last week of storage. In particular, samples T1, T2, T3, and T4 showed a significant increase in moisture content. The highest flour moisture in sample T1 might be due to the highest environmental humidity in the first week of storage (week 0), as shown in Successively, the environmental humidity in the company warehouse decreases leading to a decreasing trend in flour moisture from sample T2 to sample T6. With respect to starch content, the one-way ANOVA found that sample T0 had a significant lower starch content compared to the other samples. Moreover, the results reported in The results of temperature and humidity evolution in the warehouse of the company during flour storage are reported in With respect to the effects of storage time on dough rheological properties, p < 0.001). As shown in p = 0.044) with a high amount of variance (R2) explained by this model , consistently with earlier works [Regarding dough tenacity (P), the one-way ANOVA found a significant main effect for the factor storage time , consistent with González-Torralba et al. (2013) [2 = 0.35). In conclusion, despite González-Torralba et al. (2013) [As highlighted by Cappelli et al. (2018)  and Mast. (2013) , but wit. (2013)  found thp = 0.027) and between G and protein (p = 0.031) with a low amount of variance explained by these models . Despite the low variance explained by these models, the protein content significantly influence L and G. As reported in With respect to L and G, the one-way ANOVA did not find any statistically significant differences. However, MOLS analysis identified a statistically significant relationship between L and protein (n p = 0.0 with a lp = 0.002). In particular the samples T2 and T3 had significantly higher W values compared to the other samples (p = 0.001), protein (p = 0.020), and starch (p = 0.025), with a good amount of variance explained by the model (R2 = 0.57). Regarding flour moisture, the samples T2 and T3 had the same value (15.10), highlighting an optimum for W at this specific flour moisture content . As shown in p = 0.010), with a good amount of variance explained by the model (R2 = 0.59), consistently with an earlier work [With regard to P/L, the one-way ANOVA found a significant main effect for the factor storage time (ier work . This isp < 0.001). As shown in p = 0.003), with a high amount of variance explained by the model (R2 = 0.83).With respect to biscuit volume, the one-way ANOVA found a significant main effect for the factor storage time (As highlighted by Ma & Baik (2018) , starch p = 0.006), with a low amount of variance explained by the model (R2 = 0.36). This is consistent with the findings for the alveograph parameter P (2 = 0.83), the changes in biscuit volume seems to be mainly related to flour composition (in particular to starch content). Regarding biscuit height, the one-way ANOVA did not find any statistically significant differences. Moreover, MOLS analysis did not find any relationship both with the compositional parameters of flour  and with the environmental storage parameters (weekly mean temperature and humidity).In addition, MOLS analysis identified a statistically significant relationship between biscuit volume and weekly mean humidity , rather than from environmental storage parameters, in this short storage time. These results are supported by Shahid et al. (2019)  who founWith respect to storage time, it significantly increased dough tenacity (P) and curve configuration ratio (P/L) which were optimal at T0 (control). However, 2–3 weeks of storage highlighted a significant increase in dough strength (W), an essential alveograph parameter that is closely correlated to the technological success of leavened products. This optimum found for dough strength (W) might be considered as a great stride in understanding the effects of storage time, confirming that wheat flour reaches its optimal performances after two–three weeks of storage, in particular for W. Moreover, this information could be useful not only for biscuits production, but especially for bread and bakery products (so for the entire bakery industry).Additionally, storage time significantly affected the biscotto di Prato characteristics, highlighting the higher volume in the sample T0. However, this paper provides an additional step forward in understanding the effects of environmental storage conditions; in particular, the models developed using the MOLS analysis highlighted that dough rheology and biscuit characteristics are mainly affected by flour composition (in particular from starch content) rather than from environmental storage parameters, in the short storage period investigated. This short storage period was chosen since it reflects the approach of the majority of bread and biscuit factories which store the flours for a maximum of 6–7 weeks. However, despite a lower amount of variance explained by the models, weekly mean humidity was closely correlated with dough tenacity (P) and biscuit volume, highlighting the importance of this parameter during storage.In conclusion, the results presented here should encourage the bakery industry to carefully analyze the composition of the flours used for the production of their products to optimize the biscuit characteristics , as it is necessary to use flours with a low amount of damaged starch, as highlighted by Ma & Baik (2018) , and wit


Kirikawadi, MU51, Swarna, Shani, and Suranimala, were analyzed for proximate composition and physicochemical, functional, and antioxidant properties using standard methods. Flours from tested cassava varieties contained <1% crude fat and <2% crude protein. Flour from MU51 contained the highest amount of HCN (48.05 mg/kg) while flour from Suranimala contained the lowest (4.85 mg/kg). Total starch and amylose contents of flours were significantly lower (p < 0.05) than those of commercial wheat flour. Flour from Suranimala contained approximately similar amylopectin content as commercial wheat flour. Water absorption capacity, oil absorption capacity, water solubility index, swelling power, emulsion activity, and emulsion stability of flours from five cassava varieties were significantly higher (p < 0.05) than those of commercial wheat flour. Swarna was identified as the richest source of phenolic compounds (4.44 mmol GAE/100 g dry weight) among the five varieties. Results showed the promising application potential of flours from these five cassava varieties in different food applications such as weaning foods, bakery foods, and edible films.Cassava flour has a high potential to contribute as a raw material in the food industry. This study was aimed at characterizing flours from Sri Lankan cassava varieties with a view to explore the potential in food applications. Flours prepared from five cassava varieties, namely,  Manihot esculenta Crantz) is an important perpetual root crop that provides food for over 500 million people in the world [Cassava  for 24 h at 30°C [All five cassava varieties were cleaned and peeled. Cassava slices of 1 mm thickness were obtained from the flesh and dipped in water , redness/greenness (a∗), and yellowness/blueness (b∗) using a Chroma meter .The color was measured as lightness (4 (7.5 mL) and kept for 1 h and vortexed with distilled water (17.5 mL). After that, C6H6O (0.5 mL), conc. H2SO4 (2.5 mL), and distilled water (0.95 mL) were added and vortexed. The absorbance was measured at room temperature with a UV-Vis spectrophotometer  at 490 nm.Total starch content was determined using the method described by Alamu et al. . A 0.2 mThe size and shape of the starch granules were determined by using a scanning electron microscope (SEM) .2HCl3O2 (5 mL) separately, and 0.01 N KI (0.05 mL) was added to the solution. Blue color was read at 620 nm after 30 min, with the UV-Vis spectrophotometer . Potato amylose was used to develop the standard curve. Amylopectin content was expressed as a percentage as the deduction of amylose content of the sample from the total starch content of the same sample.Amylose content was determined by using the method described by Zhou et al. . A 20 mgThe WAC and OAC were calculated using the approach given by Sosulski et al. . One graThe WSI and SP were calculated using the approach given by Leach et al. . One graThe GT was determined as the method described by Chandra et al. . One graThe BD was calculated using the approach given by Chandra et al. . The volThe EA and ES were calculated using the approach given by Chandra et al. . In a ceExtraction of the phenolic compounds of the flour was done according to the method described by Chandrasekara and Kumar . Flour (2CO3 (10 mL) was added. It was diluted up to the 25 mL with distilled water. After incubation for 90 min at 30°C, the measurement of absorbance was done at 750 nm with the UV-Vis spectrophotometer . The TPC was expressed as mmol gallic acid equivalents (GAE)/100 g dry weight.The TPC was determined as the method described by Singleton et al. . The ext3 colorimetric method as described by Kalita et al. [3 (0.1 mL), 1 M CH3CO2K (0.1 mL), and distilled water (2.8 mL) were added. The measurement of absorbance was done at 415 nm with the UV-Vis spectrophotometer . The TFC was measured as mmol quercetin equivalents (QE)/100 g dry weight.The TFC was determined according to the AlCla et al. . The staAntioxidant activity was determined according to Kourouma et al.  with sliw/v) of C6N6FeK3 (2.5 mL). After that, the incubation of the mixture was done in a water bath  at 50°C for 20 min, and then, 10% (w/v) C2HCl3O2 (2.5 mL) was added and the centrifugation was done for 10 min at 3000 rpm. The supernatant (2.5 mL) was added to distilled water (2.5 mL) and 0.1% (w/v) FeCl3 (0.5 mL). Absorbance was measured using the UV-Vis spectrophotometer  at 700 nm.The FRAP was determined according to the method of Davies-Hoes et al. . The metp < 0.05) using Tukey's test in SPSS 16.0 software.All experiments were conducted in triplicate, and the data were expressed as mean ± standard deviation. The sample means were compared at the 95% confidence level  to 93.13% (MU51) and higher than the carbohydrate content of wheat flour  [Moisture content is an important parameter when determining the shelf life of foods. It was found that MU51 cassava flour had the lowest moisture content (4.45%) when compared with the commercial wheat flour (12.49%). It should be noted that moisture contents of cassava flours were varied with the variety. Results revealed that the moisture contents of all flour samples were less than the recommended moisture content (13% W/W)  of ediblW/W) . MoisturW/W)  (12.55%)W/W)  (13.86%)The ash content reflects the inorganic mineral content of flour samples. As shown in Shani (0.63%) and Suranimala (0.64%) varieties which were similar to the observations of Tambo et al. [Crude fat contents of studied cassava flour are lower than 1% . Accordio et al.  who obseo et al.  which leo et al. . FurtherSwarna) which were not significantly different (p > 0.05) among five cassava varieties. However, the protein contents of tested cassava flours were significantly lower (p < 0.05) than those of commercial wheat flour  to 1.7%  higher HCN contents  to 97.27 (MU51) was significantly higher (p < 0.05) than the lightness of commercial wheat flour (a∗ and b∗ of five cassava flour samples were significantly lower (p < 0.05) than a∗ and b∗ values of wheat flour. Falade et al. [b∗ in Swarna variety may be attributed by high carotene content. Gegios et al. [The color gives the first sensation to the consumers in selecting a food product. The lightness of cassava flour from five varieties that ranged from 95.50  to 63.30%  (p < 0.05) in starch contents between cassava flours could be due to genotype effect [Suranimala can be used in producing starch due to considerably higher starch contents. The functional properties of cassava starches may be improved via chemical, physical, biotechnological, and enzymatic modifications to improve the starch industry [Chemical analysis of starch revealed that starch contents of studied cassava flour vary from 38.60%  . The sige effect , maturite effect , and agre effect . Schmitze effect  have obse effect  have obse effect  is widelindustry .μm  to 15.73 μm (Shani), and results revealed that starch granule sizes of five cassava varieties were not significantly different (p > 0.05) (μm), B type granules (5-15 μm), and C type granules (<5 μm). The present observations demonstrated that studied cassava varieties contain B type granules, which are suitable for modifications such as octenylsuccinylation to form pickering emulsifiers [Concerning SEM results, we observed that clusters of cassava starch granules had smooth surfaces and truncated shapes or circular shapes with flat surfaces on one face enclosed within the parenchyma tissues . The cas > 0.05) . These flsifiers . Moreovelsifiers .Swarna cassava flour contained the highest amylose content (11.95%). The flours of all five cassava varieties contained a comparable amount of amylose (10.23-11.95%), and they were significantly lower (p < 0.05) than the amylose content of wheat flour (22.72%)  . The amyn et al.  found thn et al. , 57.Suranimala) to 308% (Kirikawadi)   and had d gruels .Swarna) and had lower OACs when compared with the commercial wheat flour  to 118.83%  than the WSI of commercial wheat flour (p > 0.05) from the SP of commercial wheat flour (The highest WSI was observed in at flour . The higat flour . The WSIp > 0.05) among the varieties but significantly higher (p < 0.05) than the GT of commercial wheat flour  and significantly lower (p < 0.05) than the BD of commercial wheat flour  EA and ES values than commercial wheat flour  (Kirikawadi) to 4.44 (Swarna) mmol GAE/100 g dry weight. However, Lima et al. [p < 0.05)  to 0.60 (MU51) mmol QE/100 g dry weight. The DRSA of selected cassava varieties was ranged from 31%  to 72% (Swarna), which was significantly (p < 0.05) the highest. The FRAP was ranged from 0.83 (Kirikawadi) to 0.93 (Swarna) mol GAE/100 g dry weight. Among tested varieties, only Swarna contained yellow-colored flesh. The highest amount of phenolic compounds was observed in Swarna relative to other four varieties. Therefore, high amounts of carotene content and phenolic compounds may be the reasons for the highest inhibition percentage in Swarna. The variation in the antioxidant content may be due to the varietal differences in cassava. The differences in genotypes highly influence the antioxidant capacity and phenolic content or root and tuber crops [Swarna, it may be suitable for functional food formulations to prevent oxidative stress-related diseases. However, further research studies are required to analyze antioxidant availability of the final products.Higher prevalence of noncommunicable diseases has gained considerable attention of antioxidant properties of foods other than their nutritional composition. Phenolics are natural antioxidant compounds, which are capable of scavenging free radicals, inhibiting oxidases, activating antioxidant enzymes, and reducing metallic ions . TPCs of < 0.05)  and ranga et al.  have obs < 0.05)  and ranger crops . In addier crops  have obsSuranimala and Shani varieties contained low amount of cyanide contents relative to the recommended levels. Water absorption capacity, oil absorption capacity, water solubility index, swelling power, emulsion activity, and emulsion stability of five flour types were significantly lower (p < 0.05) than those of wheat flour. Flour from Swarna variety demonstrated a strong antioxidant capacity and a high phenolic content compared to the other varieties. Flours from all cassava varieties contained acceptable functional properties, demonstrating the suitability to be utilized as an ingredient/raw material in different food formulations. Further, flours of these cassava varieties can be combined with the flours from other grains and legumes to develop functional flours for modern nutrimental applications.This study revealed that proximate composition and physicochemical, functional, and antioxidant properties of tested cassava flours were considerably affected by the varietal differences. The total starch contents of five varieties were lower than those of commercial wheat flour, and the major part of starch consisted of amylopectin. Flours of