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The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors., (open access.)

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified β-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-β-d-galactosamine (pNP-β-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal β-sandwich domain and a (β/α) 8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a β-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted β-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens., Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC 50 . Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron., Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections., Competing Interests: C.S., K.G. and I.B. report shareholding in Halo Therapeutics Ltd unrelated to this Correspondence. I.B. reports shareholding in Geneva Biotech SARL, unrelated to this correspondence. F.G., J.H. and I.B. report shareholding in Imophoron Ltd, related to this Correspondence. Patents and patent applications have been filed related to ADDomer vaccines and therapeutics (WO2017167988A, EP22191583.8). The other authors do not declare competing interests. ADDomer is a registered trademark of Imophoron Ltd., (© The Author(s) 2023. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4 + T cells respond to intermittent TCR stimulation more efficiently than their CD8 + T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.

PSD95-disc large-zonula occludens (PDZ) domains are globular modules of 80-90 amino acids that co-evolved with multicellularity. They commonly bind to carboxy-terminal sequences of a plethora of membrane-associated proteins and influence their trafficking and signaling. We previously built a PDZ resource (PDZome) allowing us to unveil human PDZ interactions by Yeast two-hybrid. Yet, this resource is incomplete according to the current knowledge on the human PDZ proteome. Here we built the PDZome 2.0 library for Yeast two-hybrid, based on a PDZ library manually curated from online resources. The PDZome2.0 contains 305 individual clones (266 PDZ domains in isolation and 39 tandems), for which all boundaries were designed based on available PDZ structures. Using as bait the E6 oncoprotein from HPV16, a known promiscuous PDZ interactor, we show that PDZome 2.0 outperforms the previous resource.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8 + lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity., (© 2023 Miallot et al.)

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii , each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes., (© 2022 Wiley-VCH GmbH.)

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function., (© 2022. The Author(s).)

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins., Competing Interests: Conflict of interest The University of Copenhagen has filed a patent application on the cell-based display platform. GlycoDisplay Aps, Copenhagen, Denmark, has obtained a license to the field of the patent application. Y. N. and H. C. are cofounders of GlycoDisplay Aps and hold ownerships in the company. All the other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Since the beginning of the COVID-19 pandemics, variants have emerged. Some of them display increased transmissibility and/or resistance to immune response. Most of the mutations involved in the functional adaptation are found in the receptor-binding motif (RBM), close to the interface with the receptor ACE2. We thus developed a fast molecular assay to detect mutations in the RBM coding sequence. After amplification, the amplicon is heat-denatured and hybridized with an amplicon of reference. The presence of a mutation can be detected using a mismatch-specific endonuclease and the cleavage pattern is analyzed by capillary electrophoresis. The method was validated on RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants produced in vitro before being implemented for clinical samples. The assay showed 97.8% sensitivity and 97.8% specificity. The procedure can be set up for high-throughput identification of the presence of mutations and serve as a first-line screening to select the samples for full genome sequencing., Competing Interests: The authors declare no competing interests, (© 2021 The Authors.)

Small linear motifs targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ) have been identified at the C terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A, and N showing significant interactions with dissociation constants values ranging from 3 to 82 μm. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Among the binders of the SARS-CoV-2 proteins E, 3a, or N, seven significantly affect viral replication under knock down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes., (© 2021 Federation of European Biochemical Societies.)

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science., Competing Interests: Declaration of Competing Interest AG and SJF are named inventors on patents relating to the PROSS method and various designs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity., Competing Interests: The authors have declared that no competing interests exist.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thr kinase 2 (MAST2) inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2-PDZ domain. Our strategy involved modifying the length and flexibility of the noninteracting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2-PDZ domain, with K d values decreasing from 1300 to 60 nm, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases., (© 2019 Khan et al.)

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural "bricks" to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.