A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.

Single domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, as well as therapeutic and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research - i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat IgG. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Adipokines measured during pregnancy and at birth are associated with infant negative affect.

BACKGROUND: Increased adiposity during pregnancy may be related to offspring risk for mental health disorders, although the biological mechanisms are poorly understood. One promising hypothesis is that factors secreted from adipocytes such as leptin and adiponectin may explain this association. The current study examined whether pregnancy or umbilical cord blood concentrations of leptin and/or adiponectin a) predict elevated infant negative affect at 6 months (an early life marker of risk for psychopathology); and b) help explain the association between pregnancy adiposity and increased infant negative affect. METHODS: Data came from a prospective cohort (N = 305) of pregnant individuals and their offspring. Second trimester adiposity was assessed using air displacement plethysmography. Concentrations of leptin and adiponectin were measured in second trimester plasma and umbilical cord plasma. Infant negative affect was assessed by standardized observation at 6 months. Second trimester inflammation was assessed using a comprehensive panel of cytokines. RESULTS: Lower second trimester adiponectin was associated with elevated infant negative affect, and mediated the effect of pregnancy adiposity on infant negative affect. This association was independent of the effect of second trimester inflammation. Umbilical cord leptin also predicted higher infant negative affect and mediated the association between pregnancy adiposity and infant negative affect. CONCLUSIONS: This is the first study to link pregnancy adiponectin or cord blood leptin to infant markers of risk for psychopathology, and the first to demonstrate that these adipokines mediate the association between pregnancy adiposity and offspring behavioral outcomes, suggesting novel markers of risk and potential mechanisms of effect.

The Molecular Architecture of the Nuclear Basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of Nups in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis.

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.

Evidence for Plant-Conserved Region Mediated Trimeric CESAs in Plant Cellulose Synthase Complexes.

Higher plants synthesize cellulose using membrane-bound, six-lobed cellulose synthase complexes, each lobe containing trimeric cellulose synthases (CESAs). Although molecular biology reports support heteromeric trimers composed of different isoforms, a homomeric trimer was reported for in vitro studies of the catalytic domain of CESA1 of Arabidopsis (AtCESA1CatD) and confirmed in cryoEM structures of full-length CESA8 and CESA7 of poplar and cotton, respectively. In both structures, a small portion of the plant-conserved region (P-CR) forms the only contacts between catalytic domains of the monomers. We report inter-subunit lysine-crosslinks that localize to the small P-CR, negative-stain EM structure, and modeling data for homotrimers of AtCESA1CatD. Molecular dynamics simulations for AtCESA1CatD trimers based on the CESA8 cryoEM structure were stable and dependent upon a small set of residue contacts. The results suggest that homomeric CESA trimers may be important for the synthesis of primary and secondary cell walls and identify key residues for future mutagenic studies.

Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape.

The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.

Measuring in vivo protein turnover and exchange in yeast macromolecular assemblies.

We present a comprehensive and robust protocol to track the dynamics of all proteins in a complex in yeast cells. A single member of the protein assembly is tagged and conditionally expressed, minimizing the perturbations to the protein complex. Then, SILAC labeling and affinity purification are used for the assessment of the whole protein complex dynamics. This method can determine and distinguish both subunit turnover and exchange specifically in an assembly to provide a comprehensive picture of assembly dynamics. For complete details on the use and execution of this protocol, please refer to Hakhverdyan et al. (2021).

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2.

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function.

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the holocomplex.

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

NCBP3 positively impacts mRNA biogenesis.

The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

Affinity proteomic dissection of the human nuclear cap-binding complex interactome.

A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly, is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2- and 3-related macromolecular assemblies, we have applied an affinity capture-based interactome screen where the experimental design and data processing have been modified to quantitatively identify interactome differences between targets under a range of experimental conditions. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.

LINE-1 ORF2p expression is nearly imperceptible in human cancers.

BACKGROUND: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. RESULTS: We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. CONCLUSIONS: Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex.

The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at ∼3.8 Å resolution, revealing an asymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000 Å2) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Palaeoproteomics resolves sloth relationships.

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America.

Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization.

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a 'copy-and-paste' fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles in cis with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. The following protocol describes an approach to affinity enrich ectopically expressed L1 RNPs and, using RNases, release the fraction of protein that depends upon the presence of intact RNA for retention in the immobilized macromolecules.

Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression.

The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.