Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance.

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system's molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

Asymmetric pendrin homodimer reveals its molecular mechanism as anion exchanger.

Pendrin (SLC26A4) is an anion exchanger expressed in the apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However its molecular structure has remained unknown, limiting our understanding of the structural basis of transport. Here, we determine the cryo-electron microscopy structures of mouse pendrin with symmetric and asymmetric homodimer conformations. The asymmetric homodimer consists of one inward-facing protomer and the other outward-facing protomer, representing coincident uptake and secretion- a unique state of pendrin as an electroneutral exchanger. The multiple conformations presented here provide an inverted alternate-access mechanism for anion exchange. The structural and functional data presented here disclose the properties of an anion exchange cleft and help understand the importance of disease-associated variants, which will shed light on the pendrin exchange mechanism.

Fortuitous somatic mutations during antibody evolution endow broad neutralization against SARS-CoV-2 Omicron variants.

Striking antibody evasion by emerging circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identify a clonally related antibody family from a convalescent individual. One of the members, XG005, exhibits potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members show significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface reveals how crucial somatic mutations endow XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality exhibits a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provide a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants.

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Combating the SARS-CoV-2 Omicron (BA.1) and BA.2 with potent bispecific antibodies engineered from non-Omicron neutralizing antibodies.

The highly mutated and transmissible Omicron (BA.1) and its more contagious lineage BA.2 have provoked serious concerns over their decreased sensitivity to the current COVID-19 vaccines and evasion from most anti-SARS-CoV-2 neutralizing antibodies (NAbs). In this study, we explored the possibility of combating the Omicron and BA.2 by constructing bispecific antibodies based on non-Omicron NAbs. We engineered 10 IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, 8 out of 10 bispecific antibodies showed high binding affinities to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron and BA.2, with geometric mean of 50% inhibitory concentration (GM IC50) values ranging from 4.5 ng/mL to 103.94 ng/mL, as well as the authentic BA.1.1. Six bispecific antibodies containing the cross-NAb GW01 not only neutralized Omicron and BA.2, but also neutralized the sarbecoviruses including SARS-CoV and SARS-related coronaviruses (SARSr-CoVs) RS3367 and WIV1, with GM IC50 ranging from 11.6 ng/mL to 103.9 ng/mL. Mapping analyses of 42 spike (S) variant single mutants of Omicron and BA.2 elucidated that these bispecific antibodies accommodated the S371L/F mutations, which were resistant to most of the non-Omicron NAbs. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody GW01-16L9 (FD01) in its native full-length IgG form in complex with the Omicron S trimer revealed 5 distinct trimers and one novel trimer dimer conformation. 16L9 scFv binds the receptor-binding motif (RBM), while GW01 scFv binds a epitope outside the RBM. Two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3 RBD-up conformation, improved the affinity between IgG and the Omicron RBD, induced the formation of trimer dimer, and inhibited RBD binding to ACE2. The trimer dimer conformation might induce the aggregation of virions and contribute to the neutralization ability of FD01. These novel bispecific antibodies are strong candidates for the treatment and prevention of infection with the Omicron, BA.2, VOCs, and other sarbecoviruses. Engineering bispecific antibodies based on non-Omicron NAbs could turn the majority of NAbs into a powerful arsenal to aid the battle against the pandemic.

Structural Study of SARS-CoV-2 Antibodies Identifies a Broad-Spectrum Antibody That Neutralizes the Omicron Variant by Disassembling the Spike Trimer.

The continuous emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses new challenges in the fight against the coronavirus disease 2019 (COVID-19) pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of an antibody targeting a conserved and universal epitope is urgently needed. A subset of neutralizing antibodies (NAbs) against COVID-19 from convalescent patients were isolated in our previous study. In this study, we investigated the accommodation of these NAbs to SARS-CoV-2 variants of concern (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of the SARS-CoV-2 Omicron variant. In addition, we determined the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 spike (S) protein complexed with three monoclonal antibodies targeting different epitopes, including 553-49, 553-15, and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes the virus by disassembling S trimers. IgG 553-15, an antibody that neutralizes all of the VOCs except Omicron, cross-links two S trimers to form a trimer dimer, demonstrating that 553-15 neutralizes the virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 553-49 and other antibodies targeting this highly conserved epitope as promising therapeutic reagents for COVID-19. IMPORTANCE The emergence of the Omicron strain of SARS-CoV-2 caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 neutralizing antibody, 553-49, which neutralizes all variants by targeting a completely conserved novel epitope. In addition, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by cross-linking virions and that 553-60 functions by blocking receptor binding. Comparison of different receptor binding domain (RBD) epitopes revealed that the 553-49 epitope is hidden in the S trimer and keeps a high degree of conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutic reagent against the emerging Omicron and future variants of SARS-CoV-2.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

An ultrapotent pan-ß-coronavirus lineage B (ß-CoV-B) neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope.

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes ß-coronavirus lineage B (ß-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-ß-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against ß-CoV-B and newly emerging SARS-CoV-2 variants of concern.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants.

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

The Zscan4-Tet2 Transcription Nexus Regulates Metabolic Rewiring and Enhances Proteostasis to Promote Reprogramming.

Evolutionarily conserved SCAN (named after SRE-ZBP, CTfin51, AW-1, and Number 18 cDNA)-domain-containing zinc finger transcription factors (ZSCAN) have been found in both mouse and human genomes. Zscan4 is transiently expressed during zygotic genome activation (ZGA) in preimplantation embryos and induced pluripotent stem cell (iPSC) reprogramming. However, little is known about the mechanism of Zscan4 underlying these processes of cell fate control. Here, we show that Zscan4f, a representative of ZSCAN proteins, is able to recruit Tet2 through its SCAN domain. The Zscan4f-Tet2 interaction promotes DNA demethylation and regulates the expression of target genes, particularly those encoding glycolytic enzymes and proteasome subunits. Zscan4f regulates metabolic rewiring, enhances proteasome function, and ultimately promotes iPSC generation. These results identify Zscan4f as an important partner of Tet2 in regulating target genes and promoting iPSC generation and suggest a possible and common mechanism shared by SCAN family transcription factors to recruit ten-eleven translocation (TET) DNA dioxygenases to regulate diverse cellular processes, including reprogramming.