Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function. AS is associated with functional loss of the HECT (Homologous to the E6-AP Carboxyl Terminus) E3 ligase UBE3A  encoded by the UBE3A gene2. E3 ligases are responsible for protein ubiquitination, a cellular code that dictates the destiny of most of our proteins3. The fate of the ubiquitinated protein is dependent on the number of ubiquitin (Ub) molecules conjugated and the type of Ub-Ub linkage in the poly-ubiquitin chain. Conjugation of a single Ub molecule to a lysine side chain of the target protein results in mono-ubiquitination, a modification that can change the localization or control the activity of the target. The target-attached Ub can be further modified on its N-terminus or on one of the seven lysine residues, leading to the synthesis of poly-ubiquitin chains with different linkage types. While the function of most of the Ub linkage types have not been firmly established, K48-linked Ub chains, the predominant linkage type in cells, target proteins to the proteasome for degradation (reviewed5). Although it has been well documented that UBE3A mainly synthesizes K48-linked poly-ubiquitin chains, recent evidence suggests that the UBE3A HECT domain can synthesize ubiquitin chains with different linkage type specificity6.AS is a severe neurodevelopmental disorder characterized by intellectual disability, impaired motor coordination, epilepsy and behavioral abnormalities7. Furthermore, many of the discovered UBE3A-interacting proteins do not show UBE3A-dependent proteasomal degradation, suggesting that these interacting proteins are either not bona fide UBE3A ubiquitination substrates or they receive a type of ubiquitination which does not result in their degradation.In order to understand the molecular basis of AS, several studies have been conducted to identify direct and indirect targets/interactors of UBE3A, but for most of these identified proteins their relevance for the disorder remains unknown8, and alterations in the vesicle cycle often lead to neurological disorders9. Previous studies showed an accumulation of clathrin-coated vesicles in a Ube3a mouse model of AS10, indicating a role for UBE3A in presynaptic function. However, the mechanistic underpinnings of this impaired endocytosis and its relevance to the disorder is unknown.The synaptic vesicle release cycle at the presynaptic bouton forms the basis for signal transmission between neurons11. Members of the RAB3 family, which are small GTP-binding proteins that cycle between a GTP and GDP (guanosine diphosphate)-bound state12, are key regulators of vesicle exocytosis. At least four isoforms of RAB3 have been identified: RAB3A, RAB3B, RAB3C and RAB3D. RAB3A is the most abundant isoform in the brain14 and the better-described member of the family. RAB3A has been shown to interact with members of the SNARE ) complex to facilitate vesicle fusion15. RAB3A associates with vesicles in its GTP-bound form and is released once the GTP has been hydrolyzed to GDP15. The balance between the GTP- and the GDP-bound form of RAB3A is maintained by the RAB3A GDP-dissociation inhibitor protein (GDI), which keeps RAB3A in the GDP-bound state and promotes its dissociation from synaptic vesicles16.In this study we identify the presynaptic protein Rabphilin-3A (RPH3A), as a novel binding partner of UBE3A. RPH3A is an effector protein that binds to the GTP (guanosine triphosphate)-bound form of RAB3A holding the protein in this state by preventing its GTPase activity from hydrolyzing GTP2+ binding domain of RPH3A that is highly conserved among other RAB effector proteins 11. RPH3A also contains two C2 domains homologous to the C2A and C2B of Synaptotagmin1 (Syt1), which are responsible for Ca2+ binding17. While Rab3a knock-out (KO) mice have revealed discrete but important functions of this small GTPase in vesicle exocytosis14, deletion of its effector RPH3A failed to show a major impairment18. Interestingly, mice lacking RAB3A show a marked decrease in RPH3A levels, suggesting that in the absence of RAB3A the stability of RPH3A is severely affected14.Detailed analysis of the RPH3A-RAB3A interaction has led to the identification of two regions in RPH3A that are involved in RAB3A binding: (i) specific residues in the first α-helix (α-helix a1) of RPH3A (amino-acids 45–161) and (ii) the SGAWFF sequence, a motif localized C-terminal of the Zn. UBE3A-dependent ubiquitination of RPH3A does not result in its degradation, and RPH3A levels are not changed in brains of Ube3a mice nor in Ube3a/Rab3a double mutants. Taken together, these results suggest that the ubiquitin modification of RPH3A by UBE3A serves a proteasome-independent function.Here we show that UBE3A binds to a N-terminal alpha helical segment of RPH3A and provide evidence that this binding site partially overlaps with the RAB3A binding site, the binding of which is required for RPH3A stability. We further show that RPH3A ubiquitination in mouse brain is strongly dependent on the presence of UBE3A and that RPH3A is a direct ubiquitination substrate of UBE3A in a bacterial ubiquitination assay19. One of the four high confidence interacting proteins found in this screen was Rabphilin3A (RPH3A). RPH3A is a RAB3 effector11 that contains an N-terminal zinc-finger domain and two C-terminal Ca2+-binding C2 domains  interact with UBE3A. Interestingly, RPH3A1–157 harbors the two domains essential for the interaction with the small GTPase RAB3A17: the N-terminal α-helix a1  and the SGAWFF motif, a strictly conserved motif among GTPase effectors, that is located C-terminally of the zinc finger region in RPH3A11  screenins Fig.  we were 1–157 to alanine, a mutation that has been previously described to disrupt RPH3A-RAB3A interaction21. As shown in Fig. SGAAAA was unable to associate with RAB3AQ81L, while its interaction with UBE3A was maintained, indicating that the conserved SGAWFF motif is not required for UBE3A interaction. Deletion of the N-terminal 39 amino acids (RPH3A39–157), leaving the α-helix a1 intact, did not affect either UBE3A or RAB3AQ81L binding. However, upon deletion of the complete α-helix a1 (constructs RPH3A71–157 and RPH3A87–157), the interaction with both UBE3A and RAB3AQ81L was lost. To test whether the RPH3A N-terminal α-helix is sufficient for UBE3A association, we generated RPH3A fragments harboring the α-helix a1 but lacking the entire zinc finger domain. The Y2H experiment showed that the interaction with UBE3A was completely preserved with RPH3A1–87 and only partially retained with RPH3A39–87 and RPH3A45–87. Notably, all UBE3A-RPH3A interactions appeared to be stronger with the active site mutant of UBE3A (UBE3AC817S) compared to that with wild type UBE3A, possibly suggesting increased binding in the absence of ubiquitin transfer. The molecular basis for this increased interaction with UBE3AC817S remains to be investigated. Taken together, these results indicate that RPH3A is a bona fide binding partner of UBE3A and that the minimal UBE3A binding domain of RPH3A (residues 39 to 87) includes the complete α-helix a1. Furthermore, these results show that UBE3A and RAB3AQ81L have partially overlapping binding sites on RPH3A, the exact nature of which was further investigated in this study (see below).To determine whether UBE3A and RAB3A share the same binding sites on RPH3A we first mutated the aromatic residues in the SGAWFF motif of RPH3AE. coli cells co-expressing HA-UBE3A and V5-RPH3A or HA-UBE3AC817S and V5-RPH3A, were lysed and an HA immuno-precipitation (IP) experiment was performed. Cells co-expressing HA-GFP and V5-RPH3A were used as negative control. As shown in Fig. C817S were able to pull down RPH3A, while the GFP control did not. Similarly, we investigated the ability of shorter constructs of RPH3A to interact with UBE3A. Lysates from E. coli cells co-expressing HA-UBE3A and RPH3A1–157, RPH3A39–157 or RPH3A71–157 were subjected to IP with anti-HA or IgG (control)  experiments using proteins expressed in bacteria. Specifically, ol) Fig. b. Both R19. Of note, within this small UBE3A region several AS-linked variants have been identified (https://www.ncbi.nlm.nih.gov/clinvar/?term=UBE3A[gene]). To investigate if AS-linked mutations in this region affect RPH3A binding we introduced two variants, p.Glu269Gly and p.Leu273Phe, in UBE3A by site directed mutagenesis and tested their interaction with full-length RPH3A  of UBE3A interaction. For these latter constructs the strength of interaction with RAB3A were essentially unchanged indicating that the observed loss of interaction with UBE3A is specific and not caused by changes in expression levels of the mutant proteins. Notably, based on structural analysis of the RPH3A/RAB3AQ81L complex 9 residues in the RPH3A α-helix-1 have been implicated in the RAB3A-RPH3A interaction: E50, I54, V57, I58, R60, A61, M64, E68 and R7117. Of these 9 residues we tested 7 in our yeast two-hybrid assay and confirmed for 5 of them, V57, A61, E65, E68 and R71, that they are indeed important for RAB3A binding. These results demonstrate the validity of the Y2H technique to study the effect of single amino acid changes on protein–protein interactions and indicate that the binding sites of UBE3A and RAB3AQ81L on RPH3A partially overlap.Given that the first α-helix in RPH3A is important for both UBE3A and RAB3A binding we wished to determine whether their binding sites overlap. To identify the critical residues for each interaction we performed site-directed mutagenesis of residues 55 to 72, encompassing about two-thirds of α-helix a1. To minimize the chance that the mutations would affect the α-helical structure, residues were mutated to an alanine or to a leucine , which is a variation of the Y2H assay where, in addition to two proteins fused to respectively the DNA binding domain (BD) and activation domain (AD), a third (non-fused) protein is expressed. This type of assay allows one to address if a protein interferes with or bridges the interaction between two other (DB- and AD-fused) proteins. We tested the ability of RAB3AQ81L to interfere with UBE3A-RPH3A interaction in a Y3H set-up. UBE3AC817S was used in the assay, as its strength of interaction with RPH3A is comparable to that of RAB3A with RPH3A in our Y2H. As a control, RPH3ASGAAAA mutant was used that is unable to interact with RAB3AQ81L but preserves the ability to interact with UBE3A. As shown in Fig. Q81L significantly reduced the UBE3A-RPH3A interaction. The effect of RAB3AQ81L on the UBE3A-RPH3A interaction depends on its interaction with RPH3A, as the binding between UBE3A and RPH3ASGAAAA, a RPH3A mutant that is unable to interact with RAB3AQ81L, was not reduced upon overexpression of RAB3AQ81L. Furthermore, overexpression of wild type UBE3A did not reduce the RPH3A-RAB3AQ81L interaction, suggesting that UBE3A is not able to displace the RAB3AQ81L bound to RPH3A  mice, while the RNA levels appeared to be unchanged14. This observation strongly suggests that RPH3A protein stability is dependent on the interaction with RAB3A. Given that the binding sites of RAB3A and UBE3A on RPH3A overlap, one plausible explanation for these observations is that in absence of RAB3A, i.e. in the Rab3a KO mice, UBE3A can interact with RPH3A resulting in its ubiquitination and degradation. To test this hypothesis, we deleted UBE3A in Rab3a KO mice and determined RPH3A levels in the cortex of adult mice by western blotting. Three different mutants obtained by a cross between AS mice (Ube3aE113X 22) and Rab3a KO mice  were used for this analysis: the single gene mutants Ube3a/+–/RAB3A/++ and Ube3a/++/Rab3a-/- and the double mutant Ube3a/+–/Rab3a-/-. As previously reported14, we found strongly reduced RPH3A protein levels in mice lacking both copies of the RAB3A gene (Ube3a/++/Rab3a-/-) . We found that RPH3A levels in mice lacking both UBE3A and RAB3A (Ube3a-/+/Rab3a-/-) were comparable to those observed in Ube3a/++/Rab3a-/- mice, indicating that UBE3A does not mediate the reduction in RPH3A protein levels in Rab3a KO mice.Previous studies showed that RPH3A protein levels are significantly decreased in /-) Fig. . To invebona fide ubiquitination target of UBE3A. To determine if RPH3A is ubiquitinated in vivo in an UBE3A-dependent manner we performed diGly mass spectrometry on pooled cortical tissue obtained from seven P28 Ube3a–/– mice and seven wild type littermates  cell. We first confirmed the validity of the system by co-transforming E. coli cells with UBE3A (WT or C817S), UBCH7 (the cognate E2 for UBE3A), UBA1 (rabbit E1 enzyme), ubiquitin and RING1B, a well-established target of UBE3A25. In the presence of active UBE3A and all components of the ubiquitination cascade, a smear of slower migrating bands was observed for RING1B  was present or in the absence of one of the components of the ubiquitination cascade. Target selection by UBE3A is highly specific in this assay as UBE3A failed to ubiquitinate the control protein luciferase. Importantly, we show that ARC, a protein that was suggested to be a substrate of UBE3A26 but was subsequently shown to be regulated at the transcriptional level27 is not ubiquitinated by UBE3A , we tested MDM2 as well as NEDD4 (a HECT type E3 ligase) for their ability to ubiquitinate RING1B in the bacterial ubiquitination assay. We found that neither E3 ligase ubiquitinated the UBE3A substrate RING1B, further highlighting the specificity of the system  E6 protein has been reported not only to mediate UBE3A-dependent degradation of the tumor suppressor protein p534. To determine if this type of ubiquitination can result in degradation of RPH3A in mammalian cells we used the well-characterized dihydrofolate reductase (DHFR)-ubiquitin fusion protein system27. In this system the protein of interest is fused C-terminally to DHFR-ubiquitin, resulting in the expression of a single polypeptide that is co-translationally and quantitatively cleaved by a ubiquitin-specific protease into two separate proteins: the protein of interest and DHFR-ubiquitin. By comparing the relative levels of the protein of interest and DHFR-ubiquitin, the effect of UBE3A on the stability of the target can be directly quantified. HEK293T-UBE3AKD cells in which UBE3A is partially knocked down were co-transfected with RPH3A or RING1B fused to DHFR-Ub, and either wild type UBE3A or the catalytically inactive UBE3AC817S mutant. 48 h post-transfection total protein lysates were prepared and analyzed by immunoblotting  and WT mice. As shown in Fig. Ube3a–/– mice  is often associated with proteasome-mediated degradation, mono-ubiquitination/multi-mono-ubiquitination is usually involved in protein trafficking and signalinging Fig. j. Compar30. By contrast, our interaction data indicates that the catalytically active UBE3A can bind RPH3A in the absence of E6, although the interaction is relatively weak and a stronger interaction is observed with the catalytically inactive UBE3A. Therefore, our data indicate that the UBE3A-RPH3A interaction does not require the presence of the viral E6 protein. In further support of this we recently identified the region in UBE3A required for RPH3A binding and showed that the RPH3A binding domain is located between residues 253 and 274 of UBE3A19, a region that is distant from the E6 binding site31. We now show that an AS-linked mutation in the RPH3A binding region (p.Leu273Phe) reduced the interaction with UBE3A. Collectively, our results indicate that RPH3A is a bona fide interaction partner of UBE3A.In this study we have identified the pre-synaptic protein RPH3A as a novel binding partner of UBE3A. Deletion analysis showed that an N-terminal fragment of RPH3A (residues 39–87) is necessary and sufficient to bind UBE3A. At the core of this RPH3A fragment is the α-helix a1 of RPH3A (residues 48 to 85), a region that is also involved in RAB3A binding. Alanine-scanning mutagenesis of this region revealed that the binding sites of UBE3A and RAB3A on RPH3A partially overlap, suggesting a competing binding mechanism. Consistent with this, we show in an Y3H set-up that RAB3A interferes with the UBE3A-RPH3A interaction. While this work was in progress, Martinez-Noël and colleagues also found RPH3A as an interaction partner of UBE3A in an Y3H screen with the catalytically inactive UBE3A and in the presence of the HPV E6 protein, but not when the catalytically active UBE3A was used or in the absence of E630and Supplementary Fig. Ube3a–/+) to that of wild type mice (Ube3a/++) by immunoblotting and proteomics and found no difference  levels14, suggesting enhanced breakdown of RPH3A in the absence of RAB3A. We confirmed this finding, but despite the overlapping UBE3A and RAB3A binding sites, the extreme low levels of RPH3A could not be rescued by also deleting Ube3a. This indicates that in the absence of RAB3A, RPH3A is not targeted by UBE3A for degradation. In conclusion, our data show that the UBE3A-RPH3A interaction leads to mono/multi-monoubiquitination of RPH3A, a modification that does not affect the stability of the protein neither in cells nor in the mouse brain. Mono- and multi-mono-ubiquitination have been previously described as post-translational modifications involved in chromatin remodeling and DNA repair, and mono-ubiquitination often has a role in protein signaling4. In light of this, we hypothesize that UBE3A-dependent ubiquitination of RPH3A may regulate the activity of the protein, perhaps by determining the amount of protein capable of RAB3A interaction. Further experiments will be needed to investigate this.We investigated whether RPH3A is not only a binding partner but also a ubiquitination substrate of UBE3A. We show that RPH3A is ubiquitinated in an UBE3A-dependent manner in mouse brain and is a direct target of UBE3A in a bacterial ubiquitination assay that we adapted for UBE3A target analysis. Although we observed poly-ubiquitination of well-established targets, we found that RPH3A is (multi) mono-ubiquitinated in this assay. Notably, while E6 enhances the UBE3A-RPH3A interaction . Since RPH3A is not associated with clathrin-coated vesicles35 it has been suggested that the effector plays a role at an early stage of the endocytic pathway. In the model proposed by Coppola et al.33, immediately after the dissociation from RAB3A, RPH3A is free to bind, through its N-terminus, RABAPTIN5, a known component of the endocytic machinery36. This interaction is required to initialize vesicles endocytosis leading to the assembly of the required multiprotein complex. This last aspect is particularly interesting for the Angelman Syndrome pathophysiology since Judson et al. reported that AS neurons show an impaired endocytic machinery37.RPH3A has also been implicated to play a role in endocytosis at the synaptic bouton38. This study indicated that at post-synaptic spines RPH3A acts as inhibitor of endocytosis, and by forming a ternary complex with GluN2A (a subunit of the NMDA receptor) and PSD-95, RPH3A is able to stabilize the NMDA receptor surface expression38. Different studies have reported that GluN2A expression throughout the brain is developmentally regulated with its levels in mouse brain sharply increasing from the second postnatal week40. If this finely tuned process is disturbed through mis-regulaton of RPH3A ubiquitination by UBE3A, it could potentially affect neuronal function in AS, but this has to be further investigated.Although RPH3A is known as a protein involved in the regulation of exo- and endocytosis processes at presynaptic sites, a recent study showed that RPH3A is also present in dendritic spines43, and several electrophysiological studies showed impaired synaptic function in AS mice45. Thus, in combination with the aforementioned pre and postsynaptic role of RPH3A and our findings that RPH3A is a bona fide substrate of UBE3A, the regulation of RPH3A by UBE3A could point towards an important function. However, it remains to be determined to what extent these findings are relevant to the pathophysiology of Angelman Syndrome. In particular, we recently showed that the changes in synaptic function of AS mice are also observed if only the nuclear UBE3A isoform is deleted19. This finding could suggest that the core phenotypes of AS are independent of the UBE3A-RPH3A interaction. Thus, more research is needed to address the clinical significance of our findings.UBE3A immuno-labelling has been shown to be present in the pre- and post-synaptic compartment of the synapse46. For the bacterial ubiquitination system24 and co-immunoprecipitation experiments we designed novel compatible expression vectors. pMB419 is a derivative of pCOLA-Duet1 (Novagen) that encodes a single T7 promoter followed by a lac operator, a ribosome binding site (rbs), a V5 epitope tag and a multiple cloning site (MCS). The vector has the ColA1 origin of replication and the ampicillin resistance gene. For bacterial ubiquitination experiments RPH3AFL was cloned AscI-NotI in pMB419. pYW5 is a polycistronic expression vector derived from pRSF-Duet1 (Novagen). The vector encodes a single T7 promoter followed by a lac operator, a rbs, a HA epitope tag, a multiple cloning site (MCS), a second rbs, a myc epitope and a second MCS. The vector carries the RSF origin of replication and the Spectinomycin/Streptomycin resistance gene derived from pCDF-Duet1 (Novagen). UBE3A constructs  were cloned AscI-NotI in the first MCS of pYW5. pCI-HA-NEDD4 was digested KpnI-NotI and the NEDD4 insert was cloned into AscI-NotI digested pYW5 in a three-fragment ligation using a double stranded oligonucleotide with AscI-KpnI overhangs. MDM2 (H. sapiens wild type gene) was PCR amplified from pGEX-4T-MDM2-WT, thereby introducing a 5′ AscI and 3′ NotI site. The resulting MDM2 PCR product was digested AscI-NotI and cloned into AscI-Not cut pYW5. Four derivatives of the generic plasmid pGEN4 , which co-expresses His6-tagged ubiquitin, yeast Ubc4 and rabbit E1, were constructed. In pMB277 the His6-tagged ubiquitin was replaced by wild type (untagged) ubiquitin and yeast Ubc4 by mouse UbcH7. To construct pMB278 the gene encoding wild type ubiquitin was deleted from pMB277. Deletion of the E2 in pMB277 resulted in pMB316. Similarly, deletion of the E1 in pMB277 gave rise to pMB318. The RING1BI53S construct in the dehydrofolate reductase (DHFR)-ubiquitin fusion plasmid was a generous gift of Martin Scheffner27. For bacterial ubiquitination experiments targets were cloned in the pMB419 MCS as follows. The RING1B-I53S fragment isolated from the DHFR-ubiquitin fusion plasmid was cloned BamHI-SalI, Firefly Luciferase was PCR amplified from the original construct, thereby introducing a 5′ AscI and 3′ NotI site, and cloned in AscI-NotI, and the Arc containing plasmid pRA3246 was digested AscI-NotI and cloned similarly in pMB419. For co-immunoprecipitation experiments a second pRSF-Duet1 derivative was designed. Plasmid pMB280 is a polycistronic expression vector similar to pYW5, except that it has a V5 epitope tag preceding the first MCS and a HA epitope tag preceding the second MCS. RPH3A constructs were cloned BamHI-SalI in the first MCS, generating V5-tagged proteins, while UBE3A (or GFP) was inserted in the second MCS using AscI and NotI restriction sites, introducing a HA tag at the N-terminus of UBE3A (or GFP). For some of the bacterial ubiquitination experiments pMB280 was modified to express a third gene by insertion of a double-stranded (ds)-oligonucleotide between the NotI and AvrII sites. The ds-oligonucleotide encodes a rbs followed by a Myc tag and a MCS. The resulting plasmid pMB323 can express three different proteins each with a different epitope tag  from a single T7 promoter. The HPV E6-type 16 the gene was amplified from plasmid #65 (a generous gift of Jon A Huibregtse) introducing a 5′ SbfI- and a 3′ PacI site, and cloned in the third MCS of pMB323, giving rise to pMB336. pMB336 was used to clone UBE3A  in the second MCS and the targets (RPH3A or p53 [generous gift of Jon Huibregtse]) in the first MCS. A complete list of the plasmids employed in this study can be found in Supplementary Table UBE3A constructs were amplified from mouse cDNA using a combination of forward and reverse primers as given in Supplementary Table S. cerevisiae strains used for yeast two-hybrid analysis were Y187  and Y2H Gold . Cells were grown at 28 °C in rich medium or in minimal glucose medium as previously described47.Q81L) and prey constructs (mouse RPH3A) were assessed using a mating protocol, as previously described48.Y2H interactions between bait constructs  was transformed with the bait constructs (UBE3A or RAB3A), the prey constructs (RPH3A) and a third plasmid constitutively expressing either RAB3A or RPH3A. Transformants were then selected on triple selective agar plates  and spotted as previously described47.For the yeast three-hybrid analysis E. coli BL21-GOLD (DE3) [B F–ompT hsdS(rb- mb-) dcm+Tetr gal λ(DE3) endA Hte] cells were co-transformed with bacterial expression constructs and selected on LB agar (1% (w/v) Bacto tryptone, 0.5% (w/v) Bacto yeast extract, 1% (w/v) NaCl, 1.5% agar) containing half the concentration of antibiotics as needed . Transformants were inoculated in LB medium supplemented with 2% glucose, 50 mM Tris (pH 8.0) and antibiotics as needed, and grown ON at 37 °C while shaking (200 rpm). The next morning cells were inoculated at an OD600 of 0.2 and grown at 21 °C to an OD600 of 0.7. Cells were then induced by the addition of 0.5 mM Isopropyl B-d-1-thiogalactopyranoside  and further grown at 21 °C for 16–18 h. Bacterial pellets of 20 OD600 units were lysed in 0.5 ml cold lysis buffer , RNase [0.01 mg/ml], DNase  containing 150 mM NaCl, and bound proteins were eluted by incubating the beads for 10 min at 37 °C with Laemmli sample buffer  supplemented with 50 mM DTT. Approximately 40% of the eluates (corresponding to 0.8 OD600 units) and ~ 10% of the total lysates (corresponding to 0.2 OD600 units) were loaded on NuPAGE gels, and analyzed by immuno-blotting.2. They were transfected with the respective constructs following the JetPRIME transfection system protocol (Polyplus). Total cell lysates were prepared and analysed as previously described48.HEK293T cells were grown in DMEM supplemented with 10% foetal calf serum (FCS) and 5% pen strep (PS) at 37 °C and 5% COUbe3am–/p+ mice  49 yielding both Ube3am–/p– (Ube3a KO) and Ube3am+/p+  littermates. Cortices taken from 7 P28 Ube3am–/p– mice and seven P28 Ube3am+/p+ controls were separately pooled and taken up in a lysis buffer consisting of 100 mM Tris (pH 8.5), 8 M urea and 0.15% sodium deoxycholate. Samples were transferred to the in-house proteomics facility.Mice used for the diGly mass spectrometry experiments Fig. a–c were Ube3am–/p+ mice as previously described49. Ube3atm1Alb mice were maintained (> 40 generations) in the 129S2 background (full name: 129S2/SvPasCrl) by crossing male Ube3am+/p– mice with female 129S2 wild-type mice. For immunoblotting assays, female Ube3atm1Alb (Ube3am+/p–) mice were crossed with male WT mice to obtain both Ube3am–/p+ and WT mice in the same litter.For the experiment in Fig. Ube3aE113X mice , as previously described22, crossed with Rab3a–/+mice. Ube3aE113X mice were back crossed for 13 generations in B6 background by crossing Ube3aE113X males with C57BL/6 J females. Rab3btm1Sud Rab3atm1Sud Rab3dtm1Rja Rab3ctm1Sud (Jackson lab 006375) were crossed with C57BL/6J mice to obtain Rab3b–/+Rab3a–/+Rab3d–/+Rab3c–/+. Rab3b–/+Rab3a–/+Rab3d–/+Rab3c–/+ mice were subsequently crossed once more with C57BL/6J mice to obtain Rab3b/++Rab3a–/+Rab3d/++Rab3c/++, above referred to as “Rab3a–/+”. Rab3a–/+ mice were maintained for six generations in C57BL/6J background by crossing Rab3a–/+ mice with C57BL/6J. Subsequently, Rab3a–/+ mice were crossed with Ube3aE113X mice. From the offspring, male Ube3am+/p+/Rab3a/–+ were crossed with female Ube3am+/p–/Rab3a/–+ mice, to obtain Ube3am–/p+/Rab3a/++, Ube3a/++/Rab3a-/- and Ube3am–/p+/Rab3a–/– mice used in this study.For the experiment in Fig. Mouse cortex was isolated from adult mice and lysed in 2 × Laemmli sample buffer. 20 μg of tissue lysates were loaded on SDS-PAG and analysed by UBE3A and ACTIN immunoblotting. All animal experiments were conducted in accordance with the European Commission Council Directive 2010/63/EU  and approved by the local ethics committee .23. The stoichiometry of ubiquitination was determined by relative quantitation of diGly peptides based on their mass spectral intensities.Ubiquitination sites were determined by a proteomics method based on enrichment of peptides carrying a di-Glycine (diGly) remnant as a result of trypsinization of ubiquitinated proteins, as described previouslySupplementary Information.


Synaptic cell adhesion molecule 1 (SynCAM1) also known as cell adhesion molecule 1 (CADM1) is a transmembrane cell adhesion protein that operates in a variety of physiological and pathological cellular contexts, and its interaction with the PDZ signalling protein MUPP1 have been previously implicated in autism spectrum disorder (ASD).We used in vitro pull‐down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions.So far, the investigated interaction of SynCAM1/CADM1 with MUPP1 has been mostly attributed to an unspecified region of MUPP1 PDZ domains 1–5 or exclusively to domain 2, using a yeast two‐hybrid system. We also confirmed the single interaction of native synaptosomal CADM1 with PDZ domain 2. However, in this work, using recombinant proteins overexpressed in bacteria, we found an in vitro pull‐down conditions in which all first five domains and, to a much lesser extent, MUPP1 domains 7 and 11 significantly interacted with the whole C‐terminal domain of SynCAM1/CADM1. These PDZ interactions were confirmed by a pull‐down assay using the last seven amino acids of the SynCAM1/CADM1 PDZ motif and using two fusion partners. Multiple interactions were additionally replicated using the continuous N‐terminal MUPP1 protein fragment, which included first five PDZ domains, containing either intact or mutated domain 2., representing transient low‐affinity interactions or alternative binding sites on MUPP1 when domain 2 is occupied or occluded by the interaction with other ligands. This newly identified interactions extend the potential genetic mutations, possibly affecting SynCAM1/CADM1/MUPP1 function. Possible reasons for the absence of some of the identified CADM1 PDZ interactions in mammalian extracts are discussed.We hypothesize that multiple interactions might exist in vivo SynCAM1/CADM1 C‐terminus interacts with several MUPP1 domains in overexpressed bacterial extract, while it interacts solely with MUPP1 domain 2 in mammalian cell extract. Multiple interactions might represent transient low‐affinity interactions, outcompeted in mammalian cell extract with other ligands. Newly identified interactions extent the potential targets of genetic mutations affecting SynCAM1/CADM1/MUPP1 function. The human CADM1 polyclonal antibody, against peptide VNKSDDSVIQLLNPNRQTIYFRDFRPLKD, made in rabbits (ThermoSci #PA5‐24196), corresponding to amino acids 66–94 in the extracellular Ig‐like‐V‐type domain of CADM1, was used at a 1:1,000 dilution. The sequence is identical to that of the mouse orthologue. Goat secondary horseradish peroxidase‐conjugated antibodies were purchased from Millipore. Tris (Hydroxymethyl) aminomethane (Tris) freebase, HEPES free acid, and sodium chloride (NaCl), all molecular biology grade, were purchased from Merck Chemicals.Glutathione Sepharose2.2EcoRI 5′‐ctgaattcatggcgagtgctgtgctgccgagcggatcc‐3′ and reverse SalI primer 5′‐tcaggtcgacttagatgaagtactctttcttttcttcggag‐3′. The PCR fragment was purified, digested with EcoRI/SalI, and cloned into plasmid pEGFP‐N1 (Clontech/Takara), and the DNA sequence was verified by Sanger DNA sequencing. The DNA coding region of the whole mCADM1 C terminus (amino acids R415‐I456) was obtained by PCR using forward EcoRI‐C primer 5′‐ctatgaattcagacataaaggtacatacttcactcatg‐3′ and reverse SalI primer 5′‐tcaggtcgacttagatgaagtactctttcttttcttcggag‐3′. The fragment was purified, digested EcoRI/SalI, and inserted in‐frame downstream of the cellulose‐binding domain (CBD) protein‐coding sequence into EcoRI/SalI‐digested pET34b , frame‐shifted previously by inserting single cytosine between BamHI and EcoRI sites, using site‐directed mutagenesis. Two complementary primers, 5′‐aattcgaaaagaaagagtacttcatctag‐3 and 5′‐tcgactagatgaagtactctttcttttcg‐3 (encoding the last 7 amino acids of the PDZ binding motif of mCADM1 EKKEYFI), and two additional complementary primers, 5′‐aattcgaaaagaaagagtagctgcagg‐3 and 5′‐tcgacctgcagctactctttcttttcg‐3 , were designed to additionally contain EcoRI and SalI restriction sites downstream of the stop codon. The primers were annealed and inserted into EcoRI/SalI‐digested pET34b, previously frame‐shifted by inserting a single cytosine between BamHI and EcoRI sites using site‐directed mutagenesis. The final constructs contained either the intact, 7‐amino acid‐long PDZ binding region of mCADM1 or its truncated form in‐frame with the CBD protein.Mouse CADM isoform 1 CCDS23149.1. [Q8R5M8‐1] (456 amino acid) was amplified by RT‐PCR from mouse neuroblastoma N2a cell cDNA using forward BamHI 5′‐acagggatcccatattcacccagcccacgg‐3′ primers and inserted into pET21a plasmid  digested with NdeI/BamHI. The plasmid was subsequently isolated, opened with EcoRI/SalI, and the EcoRI/SalI mCADM1 PDZ linker was inserted into these sites, resulting in‐frame fusion of the CADM PDZ motif with GlyT2N terminal protein sequences.The rat, 156‐amino acid‐long GlyT2 N‐terminal DNA fragment, previously named rGlyT2Nrcf1 . The final construct started with MUPP1‐initiating methionine and ended with the MUPP1 protein sequence YVSAKED*. To impair PDZ function of PDZ domain 2, the hydrophobic leucine in its carboxylate binding loop GLG protein sequence was replaced with arginine using site‐directed mutagenesis with primers 5′‐gatgggtccggcagagggtttggcatc‐3′ and 5′‐gatgccaaaccctctgccggacccatc‐3′.Individual MUPP1 PDZ domains fused to GST were isolated as previously described  and cultured for 1 hr in order to cool down. Following the addition of IPTG to 0.3 mM final concentration, cells were shaken overnight at 17°C. Cells were suspended in buffer A . Triton X‐100 was added to a final concentration of 1%, and proteins were released by sonication. Cell debris was removed by centrifugation. GST fusion proteins were immobilized on glutathione resin and stored at –18°C. CBD‐EKKEYFI and CBD‐EKKE fusion proteins containing cell supernatant extracts were stored at –18°C until use. The GlyT2Nrcf1‐EKKEYFI fusion protein was obtained similarly, except that IPTG induction was performed for 2 hr at 37°C. A small amount of CBD‐EKKEYFI and CBD‐EKKE, used as protein gel running controls, were isolated on microcrystalline cellulose and eluted with SDS running buffer. Since the GlyT2Nrcf1‐EKKEYFI fusion protein is highly flexible  or SDS sample buffer. Following mixing with the SDS sample buffer and a 1‐min boiling step, the samples were resolved on 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue G‐250, and protein bands were quantified by scanning and plotted with GraphPad Prism (GraphPad Software). For investigation of native CADM interactions, crude mouse brain synaptosomes were used  with the mostly disordered N‐terminal protein sequence of glycine transporter GlyT2N , which excludes identical steric constraints. Because CBD‐EKKEYFI proteins do not bind to glutathione resin (not shown), it is more likely that a weak interaction of the CBD moiety with the GST protein might partially occlude the access of glutathione.E. coli extract, in the Triton X‐100‐solubilized synaptosomal extract when the intact CADM1 protein was pulled‐down, only the interaction of MUPP1 PDZ domain 2 was confirmed using the GST‐PDZ2 fusion protein. Two major reasons can be considered when explaining the absence of an interaction in some domains in mammalian extracts containing the native CADM1 protein. The first reason might be the steric collision of an artificially introduced GST fusion tag with the intact cellular CADM1 glycosylated protein moiety . The second might be the possible competition interference of endogenous ligands. In contrast to mammalian cells extracts in bacterial systems, PDZ interactions are rare since bacterial “PDZ‐like” domains are significantly different from canonical metazoan PDZ domains (Ponting, In contrast to the pull‐down assay using Ponting, . AdditioPonting, . In contPonting, , indicatPonting, . CompetiPonting,  and randPonting, ..In overexpressed bacterial extracts, another issue should be considered, such as the high concentration of overexpressed ligand, potentially leading to false interactions of low‐affinity PDZ ligands, which might not be physiologically significant in vivoIn our case, however, the CADM1 interaction with several PDZ domains had an interaction magnitude comparable to the already established interaction of domain 2. No interaction was observed following deletion of the PDZ motif, which indicates that in bacterial extracts a high concentration of ligand allowed shorter incubation times to reach interaction equilibrium, without changing the ligand specificity. Additionally, during the trafficking and interaction in vivo, PDZ ligands are likely directly juxtaposed, without concentration equilibration with the bulk solution. This rather resembles the conditions of highly frequent availability of ligand, similarly as it is in conditions of pull‐down assay using bacterially overexpressed ligands.5Creation of the MUPP1 signalling complex may proceed via concatenation of ligands on a single molecule when the ligands occupy different PDZ domains or by clustering of MUPP1 molecules when the ligands interact with the identical MUPP1 PDZ domain. The advantage of the CADM1 interaction with multiple MUPP1 domains reported here could be the possibility of interaction with an alternative domain, when some domains are occupied with other ligands, or they are for some reasons sterically occluded. Finally, multiple low‐affinity interactions might serve as transient interaction contacts before the ligand reaches the final interaction configuration. The newly identified interaction might additionally extend the potential genetic mutations affecting SynCAM1/CADM1/MUPP1 function.The authors declare no conflicts of interest.Both authors conceived the study, collected the data, contributed to data interpretation, manuscript writing, and revisions.This article does not contain any studies involving animals or humans performed by any of the authors.


In nature, various enzymes govern diverse biochemical reactions through their specific three-dimensional structures, which have been harnessed to produce many useful bioactive compounds including clinical agents and commodity chemicals. Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are particularly unique multifunctional enzymes that display modular organization. Individual modules incorporate their own specific substrates and collaborate to assemble complex polyketides or non-ribosomal polypeptides in a linear fashion. Due to the modular properties of PKSs and NRPSs, they have been attractive rational engineering targets for novel chemical production through the predictable modification of each moiety of the complex chemical through engineering of the cognate module. Thus, individual reactions of each module could be separated as a retro-biosynthetic biopart and repurposed to new biosynthetic pathways for the production of biofuels or commodity chemicals. Despite these potentials, repurposing attempts have often failed owing to impaired catalytic activity or the production of unintended products due to incompatible protein–protein interactions between the modules and structural perturbation of the enzyme. Recent advances in the structural, computational, and synthetic tools provide more opportunities for successful repurposing. In this review, we focused on the representative strategies and examples for the repurposing of modular PKSs and NRPSs, along with their advantages and current limitations. Thereafter, synthetic biology tools and perspectives were suggested for potential further advancement, including the rational and large-scale high-throughput approaches. Ultimately, the potential diverse reactions from modular PKSs and NRPSs would be leveraged to expand the reservoir of useful chemicals. Enzymes are biosynthetic protein machineries that recognize specific substrates through unique three-dimensional structures, and catalyze the conversion of these substrates into new biomolecules . Harnessde novo biosynthetic pathway for other chemicals (Type I modular polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are prominent engineering targets due to their modular properties of enzyme assembly . Type I hemicals . This apIn this review, we briefly introduced the structure and mechanism of the modular PKS and NRPS, and thereafter focused on their repurposing examples, along with their advantages and limitations. Finally, tools in the design-build-test-learn cycle of synthetic biology and the future perspectives of the repurposing strategies were discussed.Polyketide synthases are categorized into three types, namely, types I, II, and III, according to their organization and catalytic mechanisms . Among tN-acetyltransferase-like domain rather than AT domain that recently repurposed to catalyze decarboxylation  domain modifying the degree of C- or N-methylation, formylation (F) domain, cyclization (Cy), redox-active domain (Ox and Red), among others. These steps would be repeated between modulen and modulen + 1 till the termination module. Termination module of NRPS also contains an offloading domain such as a TE domain or reductive (R) domain after T domain to release the T-bound polypeptide intermediate for the completion of NRPS biosynthesis. The TE domain hydrolyzes or cyclize the intermediate to form a linear or cyclic polypeptide. Additionally, terminal CT domain in fungal NRPSs disconnects the oligopeptide by macrocyclization, and R∗ domain mediates Dieckmann-type cyclization of PK-NRP hybrids to obtain tetramate moieties  and mutasynthesis  Table 1Table 1. udione K . Althougudione K .tcsB, resulting in modification of the FK506 C21 moiety   . Other e4 to 106 clones by iterative selection cycles and high-throughput screening has been reported. The saturation mutagenesis of three sites in the AT domain of andrimid PKS generated a library of approximately 14,000 mutants, which were analyzed via the highly sensitive LC-MS/MS screening method  could be the alternative strategy to change the specificity. In this case, the interactions between upstream ACP and downstream KS domain should be considered, including covalent interdomain linkers and non-covalent interactions between domains. By splicing at the appropriate site of the interdomain linkers, 14 KS-AT-ACP modules from 8 PKSs was successfully isolated as a functional unit, and connected to generate a total of 154 combinatorial bimodular PKSs . On the L-Tyr residue in the C-terminus of the A domain, and to assist the movement of the T domain during the catalytic cycle  domain pairs such as DD domain pairs of PKS . In addiCurrently, the favorable strategies of exchanging module specificity for the production of novel chemical derivatives seems to be; (i) KAL-AT-PAL1 unit, AT-ACP-KS unit, using heterologous DD pairs for PKS, and (ii) XU, XUC, using heterologous COM domain pairs for NRPS, respectively. Although numerous studies for altering specificity of PKS and NRPS have been reported, more engineering trials should be accumulated for various PKSs and NRPSs to optimize the strategies employed.In addition to the module specificity change, the incorporated extender unit could be further modified by various processing domains. Most of the processing target residues were the α-substituent and β-keto group of acyl-ACP intermediate of PKS, and R group of amino acid intermediate of NRPS  Table 5Table 5.In PKS, ketoreductase (KR), dehydratase (DH), and enoyl-reductase (ER) domains are the most abundant processing domains located between the AT and ACP domain as a reductive loop, which governs the degree of β-carbonyl reduction. The KR domain performs the NADPH-mediated reduction of β-keto groups to β-hydroxyl groups and determines the stereochemistry of α-substituent and β-hydroxyl group. As the full deletion of the KR domain showed loss of specificity due to impaired protein folding and stability, the inactivation of the KR domain by its key residue mutations was more effective . In anotcis-acting processing domains located in the module.Among a large variety of processing domains for non-ribosomal peptides, in this review we focused on the in situ epimerization of the α-carbon of the T domain tethered L-amino acid during peptide elongation to generate D-amino acid. One example was the exchange of E domain in module 4 of tyrocidine NRPS (ETycB) to E domain in loading module of tyrocidine NRPS (ETycA), that showed the epimerization of L-Phe to D-Phe  domain governs to D-Phe . Methylte A NRPS . A recenly lines . In anotd serine . An offlproperty .trans acting tailoring enzymes have the potential to be utilized for the production of novel derivatives but, studies are lacking. Therefore, further studies and engineering about various NRPS processing domains, including both in-cis and in-trans acting, should be quantitatively increased.Cyclization (Cy), Oxidase (Ox), and Formylation (F) domain were also expected to be the favorable targets for engineering. However, there have been no examples due to the upstream substrate specificity as well as other several mechanistic problems . BesidesTo sum up, the inactivation and swapping of the individual processing domain based on homology modeling and sequence alignment was successful, however, specialized protein–protein interactions hampered the full engineering of these domains, this required the case-by-case optimization of the swapping region.de novo enzyme assembly lines, their products were generally not the purpose of the study. Recently, technical advances of the separation and fusion of domains and modules from original enzyme assembly lines, owing to numerous engineering studies, opened the way for retro-biosynthesis. Retro-biosynthesis is a de novo pathway design that assembles reactions in a stepwise fashion in the reverse direction of synthesis, for the desired product  as the starter unit, but it has promiscuity to successfully recognize succinyl-CoA  of Aspergillus flavus  of s flavus . Based ot enzyme . This shTaxus brevifolia or the semisynthetic method, which suffered from low yields and environmentally harmful reactions, respectively. Moreover, the synthesis of the most essential precursor, amino phenylpropanoyl CoA substrates, was a major limitation as, it required protection at their amino groups before synthetic thioesterification. The authors hypothesized that A domain in tyrocidine NRPS module TycA could be used as the potential chemoselective carboxylate CoA ligase that originally had phenylalanine specificity but showed potential promiscuity. As a result, α-, β-phenylalanyl, and -phenylisoserinyl CoA were successfully generated by the A domain  to the paclitaxel biosynthesis pathway to produce various paclitaxel derivatives , 2017. AA domain . By emplA domain . These ede novo biosynthesis pathways of useful chemicals, by fusing the NRPS modules.Owing to the natural diversity of the amino acid substrates other than acyl CoA substrates, the substrate promiscuity of the NRPS modules seems to be broader than PKS modules. Although these aspects of NRPS hindered the understanding of their reactions, further advances would increase the potential of NRPS to be repurposed for the construction of de novo biosynthetic pathways for the production of useful chemicals. To overcome previous limitations of these approaches for the expansion of the diversity of their products and optimization of their productivity, the systematic strategy such as the design-build-test-learn cycle should be applied. The brief explanations, applications, and perspectives of the tools for each step are discussed below.As shown above, modular PKS and NRPS have been studied with the aim of (i) engineering for the production of novel chemicals, and (ii) repurposing the domain and module reactions toward Design tools include structural, kinetic, mechanistic, and sequence-based techniques to obtain fundamental information for the experimental design of the repurposing. Specifically, the structural and kinetic knowledge is collected and sorted to the database, and bioinformatics tools are used for domain/module selection and boundary identification based on the database. First, structural biology tools were used for the modular PKS and NRPS, which are macromolecules with numerous protein–protein interactions and dynamic conformational change during biosynthesis. Thus, the structures and interactions of the domains and one or two modules have been reported for high-resolution and dynamic scale. X-ray crystallography was the most commonly used technique that provided high-resolution images at the angstrom level . HoweverN-acetylcysteamine thioesters (acyl- or aminoacyl-SNACs) (O-sulfamoyladenosine (AMS) and adenosine vinylsulfonamide (AVS), azide-alkyne click chemistry linkers, and biotin probes were mainly used for the studies of NRPS A-T interactions  . The stul-SNACs) . The intractions . These cractions . As variBased on the information obtained from the structural and kinetic studies, computational approaches and bioinformatics would be required to design the detailed processes of the engineering of PKS and NRPS enzymes, or pathways . For exain vivo genome editing  the updated information of the protein–protein interactions and the kinetic profiles to the substrate of the enzymes, (ii) the appropriate splicing or engineering sites for the construction of synthetic parts, (iii) the rationale for the selection of compatible sets, (iv) the integration of the information of cognate reactions, sequences, substrate, and products, and (v) the reversible connections between the combination of reactions and the final product for retro-biosynthesis . Based oWe reviewed selected examples of modular PKS and NRPS repurposing for the generations of novel chemicals and pathways, followed by the roadmap in view of the synthetic biological DBTL cycle. The most important lesson from the repurposing examples was the requirement of careful considerations for dissecting the complex protein–protein interactions, despite their functionality in modular fashion. Therefore, the rational engineering design should continuously be improved by the characterization of the modular PKS and NRPS from the large amount of structural, kinetic, and genetic studies, to predict and understand the results. In parallel, the massive and rapid approaches, such as directed evolution and combinatorial strategy along with high-throughput screening, should be widely adopted to find the unpredictable factors from rational engineering. Technical advances for build tools could overcome the previous limitations for the identification of the exchangeable unit and the case-by-case optimization. Ultimately, the two approaches would complement each other by the reducing library size by rational design and the reflection of the updated information learned from the non-rational large-scale strategy. An automated pipeline of the modular PKS and NRPS repurposing for retro-biosynthesis is expected to greatly expand the reservoir of the bio-active compounds.B-KC and SH conceived the study. SH, NL, and B-KC drafted the manuscript. SH, NL, SC, BP, and B-KC contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Prunus serotine seed, was processed to produce a defatted flour (71.07 ± 2.10% yield) without hydrocyanic acid. The total protein was 50.94 ± 0.64%. According to sensory evaluation of cookies with P. serotine flour, the highest score in overall impression (6.31) was at 50% flour substitution. Its nutritional composition stood out for its protein and fiber contents 12.50% and 0.93%, respectively. Protein concentrate (PsPC) was elaborated (81.44 ± 7.74% protein) from defatted flour. Emulsifying properties of PsPC were studied in emulsions at different mass fractions; ϕ = 0.002, 0.02, 0.1, 0.2, and 0.4 through physicochemical analysis and compared with whey protein concentrate (WPC). Particle size in emulsions increased, as did oil content, and results were reflected in microscope photographs. PsPC at ϕ 0.02 showed positive results along the study, reflected in the microphotograph and emulsifying stability index (ESI) test (117.50 min). At ϕ 0.4, the lowest ESI (29.34 min), but the maximum emulsifying activity index (EAI) value (0.029 m2/g) was reached. WPC had an EAI value higher than PsPC at ϕ ≥ 0.2, but its ESI were always lower in all mass fraction values. PsPC can compete with emulsifiers as WPC and help stabilize emulsions. Nowadays, there is an increasing demand for products of high nutritional quality . ProteinProtein is available in a variety of dietary sources . In receBaked snacks, such as bread and cookies, are widely accepted and consumed throughout the world and have become an attractive target for feeding and nutritional status improvement programs. This is especially true for cookies, because they not only offer a good vehicle for protein enrichment for consumers, but also because of their wide-spread consumption (5.9 per capita in 2019) and long shelf life ,11.The implementation of wheat flour substitutes or mimicry are desirable alternatives to achieve not only a decrease in calories, but also, to obtain a healthier nutritional profile in their composition ,13. LeguAlso, food grade films, hydrogels, foams, and emulsifiers have been developed from vegetable proteins. Emulsions are capable of absorbing at the oil-in water interface or air-in water dispersion ,14. ThesIn emulsions, the emulsifying activity index, emulsifying stability index, droplet size, interfacial properties and viscosity parameters are used . Other tPrunus serotine is widely distributed, and can be found in 16 states of the Republic. Nowadays the production of the fruit goes to 467.96 tons per year [P. dulcis (19.91%) and Arachis hypogaea (22.82%), having lysine as the limiting amino acid. It has also been reported that digestibility values higher than 80% are related to an efficient amino acid bioavailability [In Mexico, the oilseed per year ,19. Howeper year . While tper year ,21. A prlability .P. serotine seed oil increase its potential as functional and nutraceutical ingredient [Its oil composition is also considered unique because of the significant content of α-eleostearic acid . This acgredient .P. serotine seed is subjected as snack, helps to not produce amygdalin because of the temperature it is subjected to. The pericarp of P. serotine accumulates amygdalin, but it is acyanogenic because it lacks enzymes to release HCN [Biotic and abiotic metabolites can contaminate crops and plant-based foods; therefore, toxins must be examined . Cyanogeease HCN . In addiease HCN . All theease HCN .However, there are some treatments that can reduce or eliminate the risk of poisoning, whereby the focus in on removal of glycoside through washing and/or pressing the food, by enzymatic breakdown of the glycoside, destroying the enzyme or a combination of these methods .P. serotine seed, two valuable products can be obtained, namely α-eleostearic acid with nutraceutical potential applications and the defatted seed with high protein content, which can be used for the development of biscuit products and concentrate protein for the stabilization of emulsions.From P. serotine oil, so we are focusing on the second product and its derivates. Therefore, the aim was to evaluate P. serotine defatted flour without hydrogen cyanide risk in cookies and protein concentrate in emulsion stability.We have previously evaluated the study of P. serotine seeds were obtained from Xochimilco’s market in Mexico City, Mexico. Wheat flour (Triticum spp.) and canola oil were purchased from a local food store in Monterrey, Nuevo Leon, Mexico. Whey protein concentrate  was food grade from Marquez Bros, International, Inc.-whey division, Hanford, CA. Solvents: hexane, n-propanol, boric acid, ethanol, phosphoric acid, and hydrochloric acid were of analytical grade . The reagents sodium chloride, sodium hydroxide, sodium azide, sodium carbonate, sodium tartrate and copper sulphate were purchased from Development of Chemical Specialities in Monterrey, Nuevo Leon, Mexico. Picric acid was from Acce Microbiology in Guadalupe, Nuevo Leon, Mexico, and Coomassie brilliant blue G-250 from ThermoFisher Scientific, Mexico. Sucrose, Tris(hydroxymethyl)aminomethane, SDS, Folin & Ciocalteu’s and bovine serum albumin (BSA) were from Sigma-Aldrich, Mexico.P. serotine (w/v) for 30 min with constant magnetic stirring, followed by scalding with hot water at 90 °C for 5 min, and drained for 7 min, followed by drying for 1 h at 60 °C in an oven with air circulation. Once dried, the oil was removed with a manual oil press  and the residue (ground seed) toasted to 100 °C for 25 min [w/v) to 25 ± 2 °C for 1 h. The ground seed was washed and filtered through Whatman paper No. 4 and dried in a hood extractor for 6 h. Finally, it was chopped in a blender and passed through a 70-mesh screen to obtain a P. serotine defatted flour [A defatted flour was elaborated from the seeds of serotine . Seeds wr 25 min . SubsequThe flour yield was determined by the following formula:P. serotine defatted flour, the methodology of Khor et al. [4,3) at 25 ± 2 °C, as Belorio et al. [Triticum spp.) was used as control.In order to measure the particle size of the r et al.  was usedo et al.  report. P. serotine defatted flour by using Association of Official Analytical Chemistry [Analysis were performed on hemistry  and compP. serotine defatted flour, hydrocyanic acid (HCN) was eliminated, a qualitative test was used according to Castro and Rodriguez [To verify that during the process of making odriguez . PicrosoP. serotine defatted flour was placed inside the jar to fill a third of it and covered quickly. The bottles were stored in the dark and after 24 h, a reading was taken. As a control, only fractionated P. serotine seeds were used (without any treatment). If the paper´s yellow color was maintained or it became light orange, there would be absolutely no problem in its consumption, but if it changed to intense orange or pink, it could only be consumed with caution and if the color became reddish or dark brown, it would not be safe to consume.The P. serotine defatted flour . Butter and sugar were mixed, then creamed with a Kitchen Aid mixer at low speed for one min. Vanilla essence and egg were added and mixed for one minute. In another bowl, all dry ingredients  were sifted and gradually added to the previous mix at low speed for 1 min and then medium speed for one min. When all the ingredients were integrated and homogenized, the dough was wrapped in plastic and allowed to cool at 4 °C for 1 h.Four variations of cookie recipe were made according to Jia et al.  with somOnce the dough had rested, it was kneaded and spread with a rolling pin and 2 × 2 cm and 0.5 cm high square cookies were cut and, placed in an aluminum tray with waxed paper to prevent them from sticking. The oven was preheated at 160 °C for 15 min and the cookies were baked for 12 min at the same temperature. After removal from the oven, the cookies were left to cool at room temperature (25 ± 2 °C).The evaluation was carried out in the Sensory Evaluation Laboratory of the Faculty of the College of Food Science at the Autonomous University of Nuevo Leon, Mexico. Fifty-five panelists (untrained) participated in the sensory test based on Jia et al. . These iP. serotine defatted flour according to the procedure described by Ramirez Pimentel et al. [v/v) 2-propanol (prolamins) and 0.1 M boric acid with 0.5% SDS pH 8 (glutelins).Proteins were extracted sequentially from l et al.  and Rayal et al. , with thw/v) was stirred for 1 h at 25 ± 2 °C. The extracts were centrifuged  at 13,000 g at 25 ± 2 °C for 20 min and the supernatants filtered (Whatman No. 4). The extraction with each solvent was repeated on the same sample sequentially and the supernatants of the three extractions were combined.The flour:solvent mixture , allowing to stand for 2 min. The samples were evaluated (microplate reader-Anthos 2020 version 2.0.5) at 620 nm.Soluble proteins were quantified from the soluble protein extractions as reported by Lopez Dellamary Toral  based onProtein patterns were analyzed according to Syros et al.  with somIn gel rails, 20 µL of each extraction of soluble protein fraction were placed (at the same previous concentrations) with distilled water. After electrophoresis, the gel was completely immersed in a fixing solution and washed three times for 10 min with distilled water. The gel was immersed and stirred in Coomassie blue dye solution (G-250) until bands were clearly evidenced.P. serotine defatted flour was determined according to the theoretical determination of proteins and other macromolecules, through zeta potential  which is the most direct characterization of the repulsion or attraction strength between their acid-base residues [w/v) at different pH with 0.1 N NaOH and 0.1 N HCl was vortexed for two minutes. Zetasizer Nano ZS90 light scattering equipment  was used. The measures were in automatic mode using a universal immersion cell  at 25 °C. The results were reported as the average of three separate injections, with three measures per injection. The averages of triplicate values were used as the values for zeta potential reported.The isoelectric point of the residues ,41. For PsPC the results obtained from the pI were taken as a basis. Variations of the procedure were undertaken to determine the one that was repeatable and had protein concentrate values ≥ 80% and ≤ 90%. All procedures were initiated by mixing the P. serotine defatted flour for 1 h in vortex with distilled water at pH 11 with 0.1 N NaOH (25 ± 2 °C), ratio 1:20 (w/v). Then the sample was isolated by centrifugation  at 13,000 g for 30 min and filtered through No. 4 Whatman paper to obtain two fractions (residue and supernatant).To obtain w/v), at two extraction times (30 min and 1 h) in vortex at 25 °C, followed by centrifugation  at 13,000 g for 30 min and filtered, again obtaining two fractions. The residue was analyzed utilizing the Kjeldhal method [In the first variation, up to two extractions of the residue obtained in the first part of the process were carried out with 5% NaCl  were tested .All final precipitates were analyzed according to proximal analysis via the Kjeldhal method based on AOAC 930.29 . The yiePsPC and whey protein concentrate) were prepared at 1% w/v in deionized water and solubilized with constant stirring. Then the sample was allowed to hydrate overnight at 4 °C. Afterwards, 0.05% sodium azide was mixed to prevent microbial growth. Different amounts of canola oil 0.1, 1, 5, 10, and 20 g were added, to obtain a variety of mass fractions . The emulsions were mixed in a homogenizer  at an initial speed of 1000 rpm for 2 min and subsequently at 3000 rpm for 3 min. All emulsions were made in triplicate and stored at 25 ± 2 °C for 18 days, and every three days, all the following analyzes were made. Whey protein concentrate (WPC) was used as a control [The emulsifying agents  at 25 ± 2 °C as Guo and Mu reports [PsPC and 1.46 for WPC, water as dispersant and an absorption of 0.1. The results were expressed as means ± standard deviation [Particle size was determined by integrated light scattering using a Mastersizer 3000 Hydro LV . The emulsions were analyzed immediately after preparation in quintuplicate. Laser diffraction measures the particle size distribution  from the angular variation of the intensity of scattered light when the laser beam passes through the particles dispersed in solution. The data are then integrated based on the angular dispersion intensity, calculating the particle size through the Mie theory of light scattering. The droplet size of emulsions was evaluated through volume-surface mean diameter  and vortexed for 5 min at 25 ± 2 °C. In a centrifuge tube, 2 mL of the solution were mixed with 7 mL of 5% sucrose solution (w/v) and all samples were centrifuged  at 3500g for 30 min at 25 ± 2 °C. Once centrifuged, three phases were observed: the oil drops in the upper phase, an intermediate phase corresponding to the 5% sucrose solution, and the aqueous phase in the lower part of the tube. The tubes were frozen at −40 °C for 24 h and then the upper layer of the oil was removed.According to Eichberg and Mokrasch  and Guo w/v) solution. To determine the concentration of adsorbed protein, 1 mL of the sample was mixed with 3 mL of an alkaline copper reagent . The samples were vigorously stirred, and allowed to rest at 25 ± 2 °C for 10 min. Subsequently, 0.3 mL of 2 N Folin-Ciocaletu was added and allowed to stand for 45 min at 25 ± 2 °C. The absorbance was immediately measured at 660 nm in a spectrophotometer  against a blank. Bovine serum albumin (BSA) was used as a standard. The interfacial protein concentration was calculated as:adC (mg/mL) is the concentration of adsorbed protein and VS is the specific interfacial area (m2/mL emulsion) of the emulsion droplets.The proteins adsorbed from the oil phase were removed by adding 20 mL of 1% SDS . Statistical comparisons were made by one-way variance analysis (ANOVA) followed by Tukey’s test using Statgraphics centurion XVII Software. The difference between means was considered significant at P. serotine defatted flour had a yield of 71.07 ± 2.10%. Its particle size  was 5.10 ± 0.03 µm, which was minor for the commercial wheat flour (Triticum spp.) with 7.31 ± 0.01 µm, making P. serotine flour 1.4 times smaller.P. serotine flour in the process of elaboration did pass through this mesh screen and its particle size was smaller than that of wheat flour (control).The AOAC 965.22  mentioneG’) correspond to flours with higher values of D4,3. Meanwhile, the biggest elastic values (G’) were found in doughs elaborated from finer flours.It is already known that the particle size of wheat flour can influence cookie quality, but this can also be true for gluten-free flours. Belorio et al.  observedP. serotine defatted flour was 4.6 times less moisture than wheat flour , the paper impregnated with picric acid changed from yellow to deep orange, which indicated that the food could be consumed, but with caution. Meanwhile, the flour with treatment paper remained with the same yellow color. This indicated that there were no potential consumption problems .P. serotine seeds has not caused any poisoning problems. In addition, the seed has been toasted for a long period of time prior to consumption.We believe that due to the low concentration of cyanogenic compounds, the consumption of P. serotine seeds.According to Alveano Aguerrebere , there iP. serotine defatted flour are shown in P. serotine defatted flour. However, the maximum amount of P. serotine defatted flour accepted by the untrained panelists in the cookies was 75% (F2) based on the value obtained in the overall acceptability category though the formulation with P. serotine defatted flour showing the highest acceptability level was F3 with a substitution of 50%.The effects of the addition of P. serotine defatted flour (F2 to F4) received a score greater than five in overall impression, making them acceptable. These cookies had greater acceptance compared to other cookies made with Californian almonds (P. dulcis), for which maximum acceptance was only 20% substitution, with a score of 5.25 in overall impression [The results obtained in the evaluation show that the cookies that were made with 75 to 25% pression .P. serotine defatted flour, those substituted by 50 and 25% (F3 and F4) were selected as a result of obtaining the highest score in overall impression  because it was preferred by the evaluators. Of the cookies made with pression .P. serotine defatted flour stood out for having fiber and for their protein content, up to 17 times higher than cookies with wheat flour (Fc), as well as, for presenting 6.57 and 2.35% lower carbohydrates, and 6.14 and 9.6% lower fat content  than control cookies. In addition, it can be said that cookies made with 50 and 25% P. serotine defatted flour have a lower gluten content compared to control cookies, since almonds are the best vegetable sources of gluten-free protein and one of the most popular ingredients in the preparation of gluten-free foods, making them a healthy alternative for people suffering from celiac disease [Cookies made with  disease .P. serotine defatted flour 16.4 ± 2.54 g soluble protein was extracted/100 mL of solution, which is equivalent to 32.15 ± 0.49% of total protein content. The soluble protein profile was albumin 76.95 ± 2.29%, globulin 13.60 ± 2.56%, glutelin 6.16 ± 0.99%, and prolamin 3.29 ± 0.37%. The relative concentration of soluble protein with respect to insoluble proteins was 3.3:1 , whereas prolamins and glutelins predominate in monocotyledonous plants . As expected of a nitrogen source, storage proteins are rich in asparagine (and aspartate), glutamine (and glutamate), and arginine , which ir et al.  the seedP. dulcis) protein, especially for polypeptides with molecular weights from 15 to 42 KDa. Typically, pepsin hydrolysis produced polypeptides with 15 to 36 KDa, followed by 15 to 20 KDa and some with 20 to 40 KDa. Therefore, P. serotine defatted flour protein may be useful in production of food protein hydrolysate and did not necessarily involve an additional process.Sze Tao and Sathe  have repK values of the stages that form and decompose the zwitterion. The point of intersection of calibration curve with the x-axis is pI value of protein [The isoelectric point of an amino acid is the pH value at which the amino acid is doubly ionized or in zwitterion concentration and is deduced from the Henderson–Hasselbach equation, as the average of the p protein ,51.P. serotine defatted flour, it was found that the specific pI for this oilseed was 3.7  ratio at pH 11 had the lowest remaining protein in the residue (13.30 ± 0.39%), which would indicate that this process allows the collection of more protein in the supernatant of the treated sample.The sample subjected to two extractions of one hour each with NaCl at a 1:20 . The resulting values were: 56.77 ± 9.20 (pH 4.5), 66.96 ± 1.21 (pH 3.7), and 72.02 ± 5.01 (pH 3.0). This was attributed to the fact that at a high ionic strength, proteins can be almost completely precipitated from their solution because of dehydration in the protein molecules, thus reducing their solubility . TherefoThe results obtained when precipitating the supernatants at pH 3.0 compared to other pH values (3.7 and 4.5) yielded a higher protein percentage. This was corroborated with the direct acidification process, which showed a concentrate value of 81.99 ± 6.96%.P. serotine defatted flour precipitate was 82.0%.The procedure was repeated two more times and results show that the treatment with direct acidification was the most effective. The average value of protein concentration in the final P. serotine seed, as well as its total and soluble protein [P. serotine protein concentrate, making this work one of the first in its findings.Researchers have identified and quantified the amino acids present in  protein ,20,40. HPsPC emulsion, the maximum droplet size value was reached at different days, but it could be considered as average on day 10. While in WPC emulsions, the range of days to reach the maximum droplet size was more stable (between days 6 and 12), the particle size was more dispersed compared to the P. serotine protein.In PsPC emulsion at ϕ 0.02 presented a droplet size of 4.39 ± 0.08 µm as a maximum, becoming the smallest and more constant emulsion during the time of experiment, and for WPC, it was at ϕ 0.002, with a value of 2.20 ± 0.20 µm. In both the control (WPC) and PsPC emulsions, on the other hand, at ϕ 0.4, the droplet size had the highest value of 6.88 ± 0.11 µm (day 9) and 14.36 ± 0.31 µm (day 15), respectively, during storage time because of coalescence.The A small droplet size is of interest in emulsion studies, because they are strongly correlated with high emulsion stability . AuthorsPsPC emulsions  to 29.34 ± 1.48 min (ϕ = 0.4) and in WPC emulsions, from 95.83 ± 7.95 (ϕ = 0.002) to 19.87 ± 1.08 min (ϕ = 0.4). For the control emulsions, less stability time was always reported compared to those of PsPC.On the contrary, the stability time diminished as the mass fraction (ϕ) increased in ESI . In PsPCw/v), the EAI values are greater, because it facilitates the formation of new droplets, and with the increase of oil, ESI value decreases. Nevertheless, at oil volumes >35% v/v, there is a marked increase in ESI, a phenomenon that has been reported also by Sun and Gunasekaran [Different authors have mentioned similar characteristics in almond proteins, wheat gluten, and acidic subunits of soy (11S globulin) ,59. Guo asekaran  for wheyEAI can be related with interfacial effect and low interaction with aqueous solutions. Our previous droplet size results can be associated with the EAI; as these indexes increased, the droplet size also increased. This can be attributed to the proteins that are surface active molecules with the capacity to improve the stability of oil-in-water emulsions, creating a protective membrane that generate repulsive interactions between oil droplets .The effect of interfacial protein concentration shows the oil drops phase separation after being centrifuged. The values suggest that emulsions were stable. When values of ϕ < 0.2 were used, it was difficult to separate the phases and reported the values.PsPC, which showed a higher value as oil volume increased, allowing more stability.The interfacial protein concentration in control emulsions was more constant, in contrast with PsPC and WPC are shown in The microphotographs in function of PsPC emulsions at their smallest mass fractions (ϕ = 0.002 and ϕ = 0.02) showed more homogenization.WPC and P. serotine seed is an important source of protein and mainly contains albumin. P. serotine defatted flour can be used to replace wheat flour at 50% in the preparation of cookies with acceptable sensory property. We believe that it is necessary to remove all cyanogenic compounds in flour of P. serotine. The Grignard test shows a positive reaction in flour without thermic treatment. The content of raw fiber in cookies was almost the same in all treatments. P. serotine flour purification can be a concentrated protein source with possible applications to stabilize emulsions.The The alkaline (pH 11) and acid (pH 3.0) process showed a higher concentration of protein than the ionic force process as a function of the sodium chloride concentration. The protein concentrate is comparable to WPC in that it forms stable emulsions with oil content of less than 20% by weight, without changing the particle size during 18 days of storage.