Malignant pleural mesothelioma (MPM) is a rare cancer of the pleura, frequently related to asbestos exposure. It is characterized by a grim prognosis, as few therapeutic options are available. Typically, MPM exhibits high molecular heterogeneity, providing opportunities for exploiting therapeutic vulnerabilities in well-defined subgroups of patients. Nevertheless, these options have been insufficiently explored. We found that inactivation of Protein Phosphatase 2A (PP2A) is a recurrent event in MPM, affecting the cancer cell phenotype and chemotherapy response. Specifically, in silico analysis showed that heterozygous loss of PTPA (PPP2R4), encoding the PP2A activator PTPA, occurred in 26

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N -terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance Statement Current colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)

An imbalance between the production and clearance of amyloid beta (Aß) has emerged as a major cause of sporadic Alzheimer’s disease (AD). Retinal wholemount studies can identify cell-specific involvement in Aß clearance mechanisms which cannot be accomplished in the brain ex vivo . Eye cross-sections of double transgenic (Tg, APP-PS1) and non-carrier sibling female mice (n = 16, 4 per group) at 3- and 9- month ages were probed with antibodies 6E10 (Aβ1-16 amino-acid residues, soluble and insoluble species), ionized calcium-binding adapter molecule 1 (IBA1, microglia/macrophage), glial fibrillary acidic protein (GFAP, astrocytes), glutamine synthetase (GS, Müller cells) and aquaporin-4 (AQP4, membrane water channel) using immunofluorescence. Wholemount neuroretinas of human AD (n = 10, mean age = 76.8, 6 females) and control (n = 10, 72.5, 5 females) donor eyes were probed with antibodies 12F4 (Aß1-42 amino-acid residues, misfolded oligomers), IBA1, GFAP, GS, AQP4 and Ulex europaeus agglutinin (UEA-l, vascular endothelium). Finally, a nanobody (soluble Aß oligomer against human Aβ1-42 peptide) conjugated with near-infrared dye (NIR-E3) was probed with 12F4 using a nanobody staining protocol in wholemount retinas. In eye cross-sections, a significant increase in 6E10, IBA1, GFAP and AQP4 was found in the older vs . younger Tg mice, suggesting Aβ accumulation resulted in gliosis and AQP4 mislocalization. In contrast, a significant reduction for 6E10 in the older vs . younger non-carrier siblings, suggests the existence of an effective Aβ clearance mechanism (e.g., macrophages and glymphatic drainage) as IBA1 and AQP4 levels were increased. In wholemount neuroretinas, macroglia degenerations (GFAP, GS and AQP4) which form the inner limiting membrane were prominent in human AD eyes, suggesting defective ocular glymphatic systems. The microglia phenotype was different from its resting stage in AD eyes, however, due to complexity of IBA1 positive resident microglia, and macrophages including monocytes, perivascular macrophages, pericytes and hyalocytes, it is difficult to define microglia phenotype, particularly activated and ameboid types between AD and control eyes. Interestingly, NIR-E3 positive perivascular macrophages (but negative to 12F4), indicating soluble Aβ clearance were predominantly found within the blood vessels of control donor eyes. This study confirms the existence of different impaired clearance mechanisms in human AD eyes.

INTRODUCTION:Impaired amyloid beta (Aβ) clearance contributes to sporadic Alzheimer's disease (AD). This study investigated retinal Aβ clearance involving neuronal, glial, and vascular interactions at the inner blood-retina barrier (iBRB), a functional analog of the blood-brain barrier (BBB). METHODS:Retinal wholemounts from AD donors and controls were analyzed alongside transgenic amyloid precursor protein/presenilin 1 (APP-PS1) and non-carrier control mouse retinal cross-sections using three- and two-dimensional ex vivo imaging. RESULTS:AD neuroretinas displayed increased larger Aβ42 deposits, microglial elongation, and substantial reductions in macroglial support and water channel expression. The uptake of soluble Aβ oligomers (SAβOs) by peripheral macrophage-like, Aβ-binding myeloid lineage cells was also diminished. In APP-PS1 mice, elevated glia levels, alongside increased APP/Aβ expression, suggest gliosis and failures in clearance processes with disease progression. DISCUSSION:Ex vivo three-dimensional retinal imaging at the iBRB provides novel insights into Aβ clearance in AD, which is difficult to replicate in ex vivo brain studies at the BBB. HIGHLIGHTS:Impaired clearance mechanisms play a key role in sporadic AD. The iBRB serves as a functional analog to the BBB. At the iBRB, the glymphatic system and microglial phagocytosis help mitigate Aβ burden. Peripheral macrophage-like myeloid lineage cells may aid SAβO clearance. The imaging plane (surface vs cross-section) may affect AD pathogenesis findings.

The imbalance between amyloid-beta (Aβ) production and clearance is a key factor in the pathogenesis of sporadic Alzheimer's disease (AD). This study investigates impaired clearance mechanisms by examining interactions among neuronal and glial cells, retinal vasculature, and blood-derived macrophages, particularly at the inner blood-retina barrier (iBRB), a functional analog of the blood-brain barrier (BBB), using wholemount neuroretinas and three-dimensional ex vivo imaging. These interactions cannot be similarly studied in brain tissues due to its highly complex structure. Wholemount neuroretinas from human AD donors ( N  = 10, mean age ± SD: 76.8 ± 9.9 years; 6 males) and controls ( N  = 10, mean age: 72.5 ± 2.2 years; 5 males), as well as eye and brain cross-sections from APP-PS1 mice and controls (3 and 9 months, all females, N = 4 per group), were analyzed using three- and two-dimensional ex vivo retinal imaging. Immunolabeling markers included Aβ1-42 peptides (12F4), soluble Aβ1-42 oligomers (SAβOs, NIR-E3 nanobody), macroglia (GFAP, GS), microglia/macrophages (IBA1), water channels (AQP4), and retinal blood vessel endothelium (UEA-1). Human AD wholemount neuroretinas exhibited a significant increase in 12F4 + Aβ deposits ( p  < 0.0001) and IBA1 + disease-associated microglia/macrophages ( p  = 0.0036), accompanied by reduced levels of macroglial markers GFAP ( p  = 0.0025), GS ( p  = 0.0015), and AQP4 ( p  = 0.0121), which are essential for glymphatic drainage, compared to age-matched controls. Clearance of SAβOs by peripheral macrophage-like monocytes through retinal blood vessels was also significantly diminished in AD retinas ( p  < 0.0001). In transgenic mouse retinal cross-sections, increased GFAP, AQP4, and IBA1 levels, along with elevated APP/Aβ peptides, indicated gliosis and AQP4 dysregulation, contributing to impaired clearance systems compared to sibling controls. The imaging plane (wholemount vs. cross-section) may influence outcomes in AD pathogenesis studies. Wholemount analysis revealed glymphatic clearance and microglial phagocytosis as compensatory mechanisms for mitigating Aβ accumulation, with peripheral macrophage-like monocytes contributing to SAβO clearance in control neuroretinas. These mechanisms were largely disrupted in AD donors. Ex vivo 3D retinal imaging, applied here for the first time to study clearance at the iBRB, provides novel insights into retinal and BBB analog processes in AD.

Clostridioides difficile is a pathogenic bacterium and a leading cause of antibiotic-associated diarrhea. Symptoms of the infection arise because of the production of large clostridial toxins that disrupt the intestinal barrier and cause an acute host inflammatory response. Epidemic C. difficile strains also produce the C. difficile transferase toxin (CDT), a binary toxin consisting of separate enzymatically active (CDTa) and cell-binding (CDTb) components. However, the role of CDT during C. difficile pathogenesis remains poorly understood. We created a CDTb nanobody (Nb) clone library and identified and purified five clones with promising CDTb-binding properties. Studies using the Carterra LSAXT platform revealed high-affinity binding interactions between the Nbs and three distinct CDTb epitopes. Functionally, these Nbs potently neutralize cellular cytopathic effects of CDT at equimolar concentrations in vitro. We further identified two distinct neutralization mechanisms-inhibition of CDTb heptamer formation and inhibition of cell surface binding, both of which are crucial for CDTa delivery into the host cell. These Nbs were used in a sandwich ELISA assay to monitor CDTb levels between 1- and 7-day post R20291 infection in the cecal material of infected mice. Notably, levels of CDTb spiked during days 3 and 4, with monomers constituting the majority of CDTb. We anticipate that these reagents will allow researchers to further expand toxin intervention and monitoring strategies to obtain a deeper understanding of the CDT mechanism of action.

Nanobodies are of high interest in many fields of medicine and biotechnology due to their high stability, tissue penetration, and engineering adaptability compared to monoclonal antibodies. However, nanobody discovery has been limited by technologies that rely on laborious library generation, panning, and clone screening techniques. Here, we demonstrate the successful adaptation of Linking B-Cell Receptor to Antigen Specificity through Sequencing (LIBRA-seq) to immunized alpacas for the rapid identification of antigen-specific nanobodies, derived from heavy-chain antibodies. We validated for nanobody discovery (nbLIBRA-seq) in two different disease settings. First, we identified over 300 antigen-specific heavy chain antibodies against human Transferrin Receptor (TfR1), also known as CD71, from a single alpaca blood sample. Experimental validation showed nbLIBRA-seq was able to identify nanobodies that exhibit specific binding to CD71, with two nanobodies also showing receptor internalization on human T cells. In a separate experiment, we tested the ability of nbLIBRA-seq to perform nanobody discovery with multiple antigens in the antigen screening library. Using fusion glycoproteins from the related respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), 1,125 antigen-specific heavy-chain expressing B cells were recovered via nbLIBRA-seq. A subset of these nanobodies was validated experimentally to possess the target antigen specificity. Together, our results illustrate the potential of nbLIBRA-seq to rapidly identify antigen-specific heavy chain antibodies for a range of diverse targets, a capability that will be of critical significance for the effective and efficient development of novel nanobody-based therapeutics against targets of biomedical significance.

Liprin-α1 is a widely expressed scaffolding protein known to regulate cellular processes such as cell motility and synaptic transmission through assembly of localized higher-order molecular complexes. However, the dynamic regulation of these complexes remains poorly understood. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins into cellular nanodomains, facilitating efficient spatiotemporal signaling. Whether liprin-α1 undergoes regulated LLPS remains unclear. Mass spectrometry-based interactomics identified PPP2R5D, the regulatory B56δ subunit of PP2A, as a liprin-α1 interaction partner via a canonical short linear motif (SLiM) in its N terminal dimerization domain. Mutation of SLiM4 nearly abolished liprin-α1 interaction with PP2A holoenzyme and resulted in a significant increase in GFP-liprin-α1 LLPS in HEK293 cells. Consistently, GFP-liprin-α1 exhibited increased droplet formation in PPP2R5D KO HEK293 cells. Phospho-analysis of liprin-α1 SLiM4 mutant via mass spectrometry revealed increased phosphorylation of multiple Ser/Thr sites, including S763, as validated by a novel phospho-specific antibody. A liprin-α1 S763E phospho-mimetic mutant appeared sufficient to drive LLPS. Expression of the PPP2R5D missense variant E420K, recurrently found in Houge-Janssens syndrome type 1 compromised suppression of liprin-α1 LLPS, correlating with increased liprin-α1 S763 phosphorylation. Mechanistically, a liprin-α1 E942A mutant unable to bind liprin-β1 underwent increased LLPS, despite preserved PPP2R5D holoenzyme binding. Furthermore, liprin-α1/β1 heterodimerization significantly decreased under conditions where liprin-α1 LLPS was promoted, i.e. upon SLiM4 or S763E mutation in WT cells, or in PPP2R5D KO and PPP2R5D E420K knock-in cells. Our findings identify liprin-β1 and PPP2R5D-PP2A as potent inhibitors of liprin-α1 LLPS, with PP2A contributing to liprin-α1/β1 heterodimerization via phosphorylation of at least liprin-α1 S763.

Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-β, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.

The Ebola filovirus (EBOV) poses a serious threat to global health and national security. Nanobodies, a type of single-domain antibody, have demonstrated promising therapeutic potential. We identified two anti-EBOV nanobodies, Nanosota-EB1 and Nanosota-EB2, which specifically target the EBOV glycoprotein (GP). Cryo-EM and biochemical data revealed that Nanosota-EB1 binds to the glycan cap of GP1, preventing its protease cleavage, while Nanosota-EB2 binds to critical membrane-fusion elements in GP2, stabilizing it in the pre-fusion state. Nanosota-EB2 is a potent neutralizer of EBOV infection in vitro and offers excellent protection in a mouse model of EBOV challenge, while Nanosota-EB1 provides moderate neutralization and protection. Nanosota-EB1 and Nanosota-EB2 are the first nanobodies shown to inhibit authentic EBOV. Combined with our newly developed structure-guided in vitro evolution approach, they lay the foundation for nanobody-based therapies against EBOV and other viruses within the ebolavirus genus.

Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, β, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1β displayed a surprising opposing function. De novo mutations in PP1β cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1β specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1β-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1β null optic nerves. Several novel important in vivo roles of PP1β in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1β lead to intellectual and developmental delays in patients.

A major challenge in antiviral antibody therapy is keeping up with the rapid evolution of viruses. Our research shows that nanobodies - single-domain antibodies derived from camelids - can be rapidly re-engineered to combat new viral strains through structure-guided in vitro evolution. Specifically, for viral mutations occurring at nanobody-binding sites, we introduce randomized amino acid sequences into nanobody residues near these mutations. We then select nanobody variants that effectively bind to the mutated viral target from a phage display library. As a proof of concept, we used this approach to adapt Nanosota-3, a nanobody originally identified to target the receptor-binding domain (RBD) of early Omicron subvariants, making it highly effective against recent Omicron subvariants. Remarkably, this adaptation process can be completed in less than two weeks, allowing drug development to keep pace with viral evolution and provide timely protection to humans.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-β oligomers (SAβOs) accumulate early, prior to amyloid plaque formation. SAβOs induce memory impairment and disrupt cognitive function independent of amyloid-β plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAβO (E3) nanobody generated from an alpaca immunized with SAβO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAβOs and amyloid-β plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAβO and amyloid-β plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAβOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAβO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.

Omicron subvariants of SARS-CoV-2 continue to pose a significant global health threat. Nanobodies, single-domain antibodies derived from camelids, are promising therapeutic tools against pandemic viruses due to their favorable properties. In this study, we identified a novel nanobody, Nanosota-9, which demonstrates high potency against a wide range of Omicron subvariants both in vitro and in a mouse model. Cryo-EM data revealed that Nanosota-9 neutralizes Omicron through a unique mechanism: two Nanosota-9 molecules crosslink two receptor-binding domains (RBDs) of the trimeric Omicron spike protein, preventing the RBDs from binding to the ACE2 receptor. This mechanism explains its strong anti-Omicron potency. Additionally, the Nanosota-9 binding epitopes on the spike protein are relatively conserved among Omicron subvariants, contributing to its broad anti-Omicron spectrum. Combined with our recently developed structure-guided in vitro evolution approach for nanobodies, Nanosota-9 has the potential to serve as the foundation for a superior anti-Omicron therapeutic.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.

enrichment associated with known signaling networks, aberrant activation of mTORC1 signaling emerged from the data collected comparing wild type and variant cells, suggesting inhibitors of mTORC1 (e.g., rapamycin) may be useful to partially restore normal function to PPP2R5D E420K variant cells.

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

ABSTRACT Nanobodies are single-domain antibodies derived from camelid animals. Here, we discovered three anti-SARS-CoV-2 nanobodies, namely, Nanosota-2, -3, and -4, from an alpaca immunized with SARS-CoV spike protein. We further characterized the antiviral activities of these Fc-tag-fused nanobodies. Notably, Nanosota-2 inhibits the prototypic SARS-CoV-2 strain in vitro (with an IC 50 of 2 pM) and in mice (at a dosage of 4 mg/kg or administered 18 hours post-challenge). These potency metrics are the best among known SARS-CoV-2 entry inhibitors. Moreover, Nanosota-3 effectively inhibits the omicron variant, both in vitro and in mice, regardless of the administration route (intraperitoneal or intranasal). Furthermore, Nanosota-3 has been biochemically engineered to inhibit both early and currently circulating subvariants of omicron. Additionally, Nanosota-4 uniquely inhibits both SARS-CoV-1 and SARS-CoV-2. Cryo-EM data revealed that the three nanobodies bind to functionally critical and non-overlapping regions in the spike protein. Given their cost-effectiveness, ease of adaptation to new viral strains, and potential use as inhalers, the Nanosota series are powerful therapeutic tools against coronavirus pandemics. IMPORTANCE The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics.