Biology subjects

Sutton, R. B.

Publications and source records attributed to Sutton, R. B..

9 recordsLinked to original sources

bioRxiv2026

Amyloid/RNA Condensates with Germ Granule Characteristics Are Present in Rat Epididymal Spermatozoa and Exhibit Prion-Like Propagation Activity

Environmental exposures to the germline can alter its epigenome resulting in new traits being acquired by offspring. While the epigenetic carrier in mammalian spermatozoa remains unknown, studies in C.elegans show that amyloids and sperm-specific populations of germ granules, phase-separated biomolecular condensates enriched in RNA-binding proteins and small noncoding RNAs, transmit heritable epigenetic phenotypes to descendant cells. Here we discovered that rat epididymal spermatozoa contain germ granule-like condensates in the head perforatorium, a cytoplasmic derived structure that participates in early post-fertilization events. The perforatoria condensates contain evolutionarily conserved germ granule markers and small noncoding RNAs and are enriched in amyloid, including those of the clinically relevant amyloidogenic proteins androgen receptor (AR) and TDP43. Further, perforatoria extracts exhibit prion-like propagation behavior by functioning as molecular seeds to template amyloid assembly in recombinant AR and TDP43. We propose that nature has conserved sperm germ granule-like structures throughout evolution to allow an organism to respond and adapt to a changed environment by passing environmentally encoded information to the embryo via a non-Mendelian protein-based mechanism of inheritance.

cell biology↗

bioRxiv2023

The Mouse Epididymal Amyloid Matrix: A Mammalian Counterpart of a Bacterial Biofilm

The mouse epididymis is a long tubule connecting the testis to the vas deferens. Its primary functions are to mature spermatozoa into motile and fertile cells and to protect them from pathogens that ascend the male tract. We previously demonstrated that a functional extracellular amyloid matrix surrounds spermatozoa in the epididymal lumen and has host defense functions; properties not unlike that of an extracellular biofilm that surrounds and protects a bacterial community. Here we show the epididymal amyloid matrix also structurally resembles a biofilm by containing eDNA, eRNA, and mucin-like polysaccharides. Further these structural components exhibit comparable behaviors and perform functions like their counterparts in bacterial biofilms. Our studies suggest that nature has used the ancient building blocks of bacterial biofilms to form an analogous structure that nurtures and protects the mammalian male germline.

cell biology↗

bioRxiv2023

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene (DYSF). This disease has two known pathogenic missense mutations that occur within dysferlins C2A domain, namely C2AW52R and C2AV67D. Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlins ability to repair cells. The failure of C2AW52R and C2AV67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes {beta}-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1{beta}. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2AW52R and C2AV67D, as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

biophysics↗

bioRxiv2023

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

immunology↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗

bioRxiv2022

Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

Ferlins are complex, multidomain proteins whose functions involve membrane fusion, membrane repair, and exocytosis. Progress in studying the domain composition of ferlins has been problematic due to the large size of the ferlin proteins and the difficulty in computing accurate residue ranges for each respective ferlin protein domain. However, recent advances in in silico protein folding methods have significantly enhanced the understanding of the domain structure of these complex proteins. To compute unbiased domain boundaries, we used RoseTTAFold to assemble full-length models for each of the six human ferlin proteins (dysferlin, myoferlin, otoferlin, Fer1L4, Fer1L5, and Fer1L6). Despite the differences in amino acid sequence between the ferlin proteins, the domain ranges and the distinct subdomains in some of the ferlin domains were remarkably consistent. Further, the RoseTTAFold/AlphaFold2 in silico boundary prediction methods allowed us to describe and characterize a previously unknown C2 domain, ubiquitous in all human ferlins, which we refer to as C2-FerA. At present, the ferlin domain-domain interactions implied by the full-length in silico models are predicted to have a low accuracy; however, the use of RoseTTAFold and AlphaFold2 as a domain finder has proven to be a powerful research tool in understanding ferlin structure.

biophysics↗

bioRxiv2022

Structural features of Dnase1L3 responsible for serum antigen clearance

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 does not rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are several key differences between the two enzymes. Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. The CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA to prevent autoimmune pathology. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.

biochemistry↗

bioRxiv2021

Tumor-Antagonizing Fibroblasts Secrete Prolargin as Tumor Suppressor in Hepatocellular Carcinoma

Systemic treatment of hepatocellular carcinoma (HCC) targets the tumor microenvironment (TME) by combining immunotherapy with angiogenesis inhibitors. Cancer-associated fibroblasts (CAF) are important components of the TME, however their targeting in clinics remains challenging. To this end, the existence of tumor supressing CAF and their poor characterisation is a major conundrum. Starting from proteomics and single cell analysis, we outline CAF heterogeneity in HCC and describe a subtype of CAF that express a novel tumor-related protein prolargin. Upon secretion prolargin is deposited in the TME where its levels positively correlate with patient outcome (HR=0.37; p=0.01). In vivo, tumors with lower prolargin expression display faster progression (5-fold; p=0.01) and stronger angiogenesis. Mechanistically, aggressive HCC cells degrade prolargin using matrix metalloprotease 3 (MMP3). We show that prolargin binds and inhibits several growth factors, key to tumor progression. Inhibiting prolargin degradation combined with sorafenib, demonstrated superior tumor control compared to sorafenib treatment alone. In conclusion, prolargin-expressing CAF have tumor-antagonizing properties. Stabilizing prolargin tumoral levels should be considered for systemic therapy of HCC, involving CAF in existing TME targeting.

cancer biology↗

bioRxiv2018

Dynamic Control of Ca2+ Binding in the C2 Domains of Synaptotagmin 1

Synaptotagmin senses fluctuations in the Ca2+ environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+ at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+ -binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+ binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+ -binding pockets of C2A and C2B. The volume of the unbound Ca2+ -binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+ -binding pocket in C2A. C2B maintains a more compact Ca2+ -binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+ binding loops would likely affect Ca2+ affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+ -binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+ -binding pocket and provides an explanation for the observed lethal phenotype.

bioinformatics↗