Biology subjects

Sutherland, L.

Publications and source records attributed to Sutherland, L..

11 recordsLinked to original sources

bioRxiv2026

Impact of RNA modification and IL-12 boosts on mRNA-LNP vaccine-elicited CD8+ T cell responses

Development of effective HIV-1 vaccines requires strategies for inducing both CD8+ T cell and neutralizing antibody responses. Nucleoside-modified mRNA-lipid-nanoparticles (LNP) induce more potent antibody responses than unmodified mRNA-LNP vaccines, while unmodified mRNA is typically employed for CD8+ T cell-inducing vaccines. Here, we compared the abilities of differing ratios of unmodified versus modified mRNA expressing a SIV conserved-region CD8+ T cell immunogen co-administered with IL-12-encoding mRNA-LNP during boosting to induce CD8+ T cell and humoral responses in non-human primates, assessing responses in ChAdOx1-primed controls in parallel. Serial priming and boosting with fully-unmodified mRNA elicited higher-magnitude, more CD8-biased T cell responses than fully-modified mRNA, that were durably maintained after the IL-12-encoding mRNA boosts. Inclusion of 30% nucleoside-modified mRNA enhanced antibody titers, suggesting potential benefits to a mixed-composition vaccine.

immunology↗

bioRxiv2026

Rapid vaccine induction of macaque HIV-1 V2 Apex broadly neutralizing antibodies with immunogenetic signatures that are potentially translatable to humans

Due to the complex biology of HIV-1 broadly neutralizing antibodies (bnAbs), HIV-1 vaccines must target unmutated germline precursor antibodies with B cell receptors that have the appropriate specificity and genetic features to develop neutralization breadth. Previous work has shown that immunization or infection in macaques primes unmutated precursor antibodies that target the broadly neutralizing epitope composed of the second variable region and proximal glycans (termed V2-apex site) of HIV-1 envelope (Env). Macaque V2-apex bnAb precursors elicited thus far have homogeneous usage of the second reading frame of a diversity (D) gene found only in macaques, calling into question whether HIV-1 Env immunogens can elicit only this canonical, macaque-specific V2-apex antibody response, limiting human vaccination success. Here, we show that vaccination of rhesus macaques with Env conjugated to a ferritin nanoparticle (OPT4-scNP) rapidly elicited V2-apex bnAbs with distinct D gene segment usage and motifs that are not restricted only to macaques. After two priming immunizations, V2-apex antibodies with non-canonical HCDR3s exhibited greater neutralization potency and breadth than V2-apex antibodies with previously observed HCDR3 motifs. Following sequential boosting immunizations, a V2-apex bnAb lineage with a novel HCDR3-encoded EDGED motif increased its neutralization breadth, including neutralization of viral isolates bearing the N130 glycan, which shields the V2-apex from recognition. High-resolution structures of two members of this V2-apex bnAb lineage, called DH2050, defined a novel binding mode in which it used its EDGED motif to contact the C-strand peptide and the N160 glycan within the V2-apex site. Altogether, we define a novel bnAb binding mode to the V2 Apex and demonstrate that HIV-1 Env sequential vaccination elicits V2-apex bnAbs with immunogenetic signatures that are potentially translatable to the human immune system.

immunology↗

bioRxiv2025

A simple method for mapping the location of cross-β forming regions within protein domains of low sequence complexity

Protein domains of low sequence complexity are unable to fold into stable, three-dimensional structures. In test tube studies, these unusual polypeptide regions can self-associate in a manner causing phase separation from aqueous solution. This form of protein:protein interaction has been implicated in numerous examples of dynamic morphological organization within eukaryotic cells. In several cases, the basis for low complexity domain (LCD) self-association and phase separation has been traced to the formation of labile cross-{beta} structures. The primary energetic force favoring formation of these transient and reversible structures is enabled by polypeptide backbone interactions. Short, contiguous networks of peptide backbone amino groups and carbonyl oxygens are zippered together intermolecularly by hydrogen bonding as described by Linus Pauling seven decades ago. Here we describe a simple, molecular biological method useful for the identification of localized, self-associating regions within larger protein domains of low sequence complexity. SignificanceThis study describes a molecular biological method for analyzing protein domains of low sequence complexity in search of segments that mediate self-association and consequent phase separation both in vitro and in vivo. Small regions allowing for self-association correspond to sequences that specify the formation of labile cross-{beta} structural order. When juxtaposed to the C-terminus of GFP, cross-{beta} prone regions suppress fluorescence. A tiled scan of overlapping fragments of the low complexity domain (LCD) of the TDP-43 RNA binding protein pinpointed an evolutionarily conserved sequence of twenty amino acids essential for self-association, phase separation and the formation of nuclear speckles. The screening method described herein should be useful for the analysis of any LCD believed to function via homotypic self-association.

biochemistry↗

bioRxiv2023

p75NTR neurotrophin receptor function is redundant for development, growth and fertility in the rat.

The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-{Delta} alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. Thus, p75NTR function appears largely dispensable for normal development, growth and basal homeostasis in the rat. The availability of constitutive and conditional p75NTREx2-{Delta} rats should, however, provide new opportunities to investigate specific roles of p75NTR upon injury and during regeneration.

genetics↗

bioRxiv2023

Oxidative regulation of TDP-43 self-association by a beta-to-alpha conformational switch

An evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an -helical or {beta}-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-{beta} structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-{beta} structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of -helical structure in precisely the same region formerly functioning to facilitate {beta}-strand mediated self-association. We further observe that the -helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the {beta}-to- conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains. Significance StatementThe TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-{beta} structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an -helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.

biochemistry↗

bioRxiv2023

Vaccine induction of CD4-mimicking broadly neutralizing antibody precursors in macaques

The CD4 binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the CD4bs is occluded by glycans, immunogen expansion of appropriate naive B cells, and selection of functional antibody mutations. Here, we demonstrate immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAbs bound to HIV-1 Env demonstrated binding angles similar to human bnAbs and heavy chain second complementarity determining region-dependent binding characteristic of all known human CD4-mimicking bnAbs. Macaque nAbs were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAbs. This vaccine study initiated the B cells from which derive CD4bs bnAbs in primates, accomplishing the key first step in development of an effective HIV-1 vaccine.

immunology↗

bioRxiv2021

Functional Anatomy of the TDP-43 Redox Sensor

TAR binding protein 43 (TDP-43) is an RNA binding protein that assists in the maturation, export and sub-cellular localization of mRNA. The carboxyl terminal 153 residues of TDP-43 are of low sequence complexity and allow for self-association of the protein in a manner leading to its phase separation from an aqueous environment. These interactions assist TDP-43 in forming cytoplasmic RNA granules involved in the transport of mRNA for localized translation. Self-association of the TDP-43 low complexity (LC) domain is facilitated by a region of twenty five residues that are of extreme evolutionary conservation. The molecular basis for self-adherence of the protein through this region has been illuminated by a combination of structural and biochemical studies, allowing definition of a morphologically specific cross-{beta} structure predicted to be weakly assembled by main chain hydrogen bonds. In this study we have investigated the importance of individual, Pauling hydrogen bonds hypothesized to facilitate self-adherence of the TDP-43 LC domain.

biochemistry↗

bioRxiv2021

Tracing the genetics of neurological disease to the mutation-directed addition of single hydrogen bonds

Mutations causative of neurological and neurodegenerative disease can occur in coding regions that specify protein domains of low sequence complexity. These autosomal dominant mutations can be idiosyncratic in their recurrent appearance at the same amino acid. Here we report studies of recurrent mutations in proline residues located within low complexity (LC) domains associated with the neurofilament light chain protein, the microtubule-associated tau protein, and the heterogeneous nuclear RNPA2 protein. All such mutations manifest their effects by directing formation of variant proteins endowed with the addition of a single, main chain hydrogen bond specified by the variant amino acid replacing proline. Here we show that methylation of the peptide backbone nitrogen atom associated with these variant amino acids eliminates the aberrant hydrogen bond and restores normal protein function.

biochemistry↗

bioRxiv2021

Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages from livestock. Porcine and bovine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages. Pig PSCdMs were productively infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and African Swine Fever Virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, Pig PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied in parental stem cells, or directly by lentiviral vector transduction. PSCs and differentiated derivatives therefore provide a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in livestock.

immunology↗

bioRxiv2020

Dynamic structural order of a low complexity domain facilitates assembly of intermediate filaments

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent upon either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1 IFs functions by forming labile cross-{beta} interactions. These interactions are seen in polymers made from the tail domain alone as well as assembled IFs formed by the intact Tm1 protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology. One Sentence SummaryA new form of protein folding that interconverts between the structured and unstructured states controls assembly of intermediate filaments.

biochemistry↗

bioRxiv2020

Transiently structured head domains control intermediate filament assembly

Low complexity (LC) head domains 92 and 108 residues in length are, respectfully, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, {beta}-strand-enriched polymers. Solid state nuclear magnetic resonance (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for {beta}-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease causing mutations reciprocally alter NFL and desmin head domain self-association, yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, {beta}-strand-enriched self-interactions may broadly influence cell morphology.

biochemistry↗