Biology subjects

Rout, M. P.

Publications and source records attributed to Rout, M. P..

15 recordsLinked to original sources

bioRxiv2026

Multivalent Anti-ACE2 Nanobodies Confer Broad Pan-Sarbecovirus Protection

The continual emergence of SARS-CoV-2 variants that rapidly evade conventional spike-directed neutralizing antibodies, together with the ongoing risk of cross-species spillover and new sarbecovirus outbreaks, underscores the need to develop broadly acting, escape-resistant therapeutic agents. Here, we optimized a nanobody discovery pipeline incorporating competition-based yeast surface display assays to isolate single-chain variable heavy chain-only antibody domains (VHHs or nanobodies) that bind human ACE2 and inhibit SARS-CoV-2 entry. Dimeric VHHs, as well as bivalent and tetravalent Fc-fusion proteins exhibited markedly increased antiviral activity, blocking a broad panel of SARS-CoV-2 variants and diverse sarbecoviruses at low-nanomolar to picomolar concentrations. These agents did not affect ACE2 enzymatic function or cell surface expression. The VHH-Fc fusion proteins had favorable pharmacokinetics and conferred prophylactic protection in mouse models of both SARS-CoV-2 and SARS-CoV infection, showcasing their potential as broadly acting receptor-targeted biologics against pandemic-threat viruses.

immunology↗

bioRxiv2024

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4s release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

biophysics↗

bioRxiv2024

The Molecular Architecture of the Nuclear Basket

The nuclear pore complex (NPC) is the sole mediator of nucle-ocytoplasmic 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 NPCs 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

biophysics↗

bioRxiv2024

Integrative spatiotemporal map of nucleocytoplasmic transport

Nuclear Pore Complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargos, including pre-ribosomes and viral capsids. It quantifies how flexible phenylalanine-glycine (FG) repeat proteins raise an entropy barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by "fuzzy" multivalent interactions, redundant FG repeats, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

cell biology↗

bioRxiv2023

Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.

Nanobodies are single domain antibody variants that bind an antigen with the precision and affinity of a conventional antibody at only a fraction of their size. In solving the crystal structures of our nanobody-GFP complexes and compared with other available structures, we uncover mechanism that enable nanobodies to function so efficiently and effectively as single-domain antibodies. We show that unlike conventional antibodies, a nanobody repertoire maximizes sampling of their antigen surface by binding a single antigen in at least three different orientations which can be predicted by their paratope composition. We also structurally reengineering these nanobodies to improve their antigen affinity, their stability, or both - results which also revealed the strong connection between nanobody stability and affinity. We achieved this by either directly modifying the paratope, or by altering a particular region within their third framework, which is a highly conserved area that we determined plays a role in controlling nanobody stability. Our study suggests that these unique characteristics of nanobodies allow them to interact with antigens as effectively as conventional antibodies, despite their smaller size. This understanding provides methods to facilitate optimizing, humanizing and functionalizing nanobodies, thus paving the way for their utilization in diverse areas such as research, diagnostics, and therapeutic development. Significance StatementNanobodies are a unique type of antibody fragment found in select animals, containing all its antigen binding ability reduced to a single [~]15 kDa protein. There is increasing development of nanobodies for research, diagnostics, and therapeutics, yet how nanobodies function so effectively as single domain antigen binders with the precision and affinity of conventional antibodies is unclear. In this study, we present key observations to help answer this question, where one key finding is the strong relationship between nanobody stability and antigen affinity aided by the identification of a highly conserved region in nanobodies essential for maintaining nanobody stability. This region may have been retained in nanobodies in lieu of stabilizing mechanisms induced by dimerization as seen in conventional antibodies.

biochemistry↗

bioRxiv2023

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

biochemistry↗

bioRxiv2023

Dynamic molecular mechanism of the nuclear pore complex permeability barrier

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

biophysics↗

bioRxiv2022

Mex67 paralogs mediate division of labor in trypanosome RNA processing and export.

In opistokhonts (animals and fungi), mRNA export to the cytoplasm is mediated by the Mex67/Mtr2 (NXF1/NXT1) heterodimer via the nuclear pore complex (NPC). In contrast to most nucleocytoplasmic transport, mRNA export requires ATP-dependent remodeling machinery, and in animals and fungi is Ran-independent. While most eukaryotes possess one Mex67 gene, trypanosomes have three distinct Mex67 paralogs, while retaining a single Mtr2 gene. We show here that these paralogs, TbMex67, TbMex67b and TbMex67L, have differing and non-redundant roles in RNA export. Specifically, TbMex67 and TbMex67b retain a canonical role in mRNA export, albeit associating with specific mRNA cohorts, but in contrast, TbMex67L is primarily involved in ribosome biogenesis. Together with the association of all Mex67 paralogs with the Ran machinery, these findings indicate significant departures in RNA export mechanisms in these divergent organisms, with implications for evolutionary origins and diversity in control of gene expression.

cell biology↗

bioRxiv2022

The nucleoporin Nup170 mediates subtelomeric gene silencing through the Ctf18-RFC complex and PCNA

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interaction, genetic interaction, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170{Delta}. The NPC therefore mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

cell biology↗

bioRxiv2021

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.

biochemistry↗

bioRxiv2020

Heh2/Man1 may be an evolutionarily conserved sensor of NPC assembly state

Integral membrane proteins of the Lap2-emerin-MAN1 (LEM) family have emerged as important components of the inner nuclear membrane (INM) required for the functional and physical integrity of the nuclear envelope. However, like many INM proteins, there is limited understanding of the biochemical interaction networks that enable LEM protein function. Here, we show that Heh2/Man1 can be affinity purified with major scaffold components of the nuclear pore complex (NPC), specifically the inner ring complex, in evolutionarily distant yeasts. Interactions between Heh2 and nucleoporins is mediated by its C-terminal winged-helix (WH) domain and are distinct from interactions required for INM targeting. Disrupting interactions between Heh2 and the NPC leads to NPC clustering. Interestingly, Heh2s association with NPCs can also be broken by knocking out Nup133, a component of the outer ring that does not physically interact with Heh2. Thus, Heh2s association with NPCs depends on the structural integrity of both major NPC scaffold complexes. We propose a model in which Heh2 acts as a sensor of NPC assembly state, which may be important for NPC quality control mechanisms and the segregation of NPCs during cell division.

cell biology↗

bioRxiv2020

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). The CBC has come under renewed investigative interest in recent years due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome - including the proteins SRRT (a.k.a. ARS2) and ZC3H18, and macromolecular assemblies such as the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative, non-canonical 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, including their intersections and differences, we have applied an affinity capture-based interactome screening approach, where the experimental design and data processing have been modified and updated to identify interactome differences between targets under a range of experimental conditions, in the context of label-free quantitative mass spectrometry. 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.

biochemistry↗

bioRxiv2018

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

Protein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

biophysics↗

bioRxiv2017

Rapid isolation of functionally intact nuclei from the yeast Saccharomyces

Most available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmatic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.

cell biology↗

bioRxiv2017

Dissection of purified LINE-1 reveals distinct nuclear and cytoplasmic intermediates

1.Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications and quantitative mass spectrometry, we have characterized the proteins associated with distinctive L1 macromolecular complexes. Our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in template-primed reverse transcription.

molecular biology↗