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Background The only known genetic cause of congenital stationary night blindness (CSNB) in horses is a 1378 bp insertion in TRPM1. However, an affected Tennessee Walking Horse was found to have no copies of this variant. Objectives To identify the genetic cause for CSNB in an affected Tennessee Walking Horse. Study design Case report detailing a whole-genome sequencing (WGS) approach to identify a causal variant. Methods A complete ophthalmic exam, including an electroretinogram (ERG), was performed on suspected CSNB-affected horse. WGS data were generated from the case and compared with data from seven other breeds (n = 29). One hundred candidate genes were evaluated for coding variants homozygous in the case and absent in all other horses. Protein modelling was used to assess the functional effects of the identified variant. A random cohort of 90 unrelated Tennessee Walking Horses and 273 horses from additional breeds were screened to estimate allele frequency of the GRM6 variant. Results ERG results were consistent with CSNB. WGS analysis identified a missense mutation in metabotropic glutamate receptor 6 (GRM6) (c.533C>T p.Thr178Met). This single nucleotide polymorphism (SNP) is predicted to be deleterious and protein modelling supports impaired binding of the neurotransmitter glutamate. This variant was not detected in 273 horses from three additional breeds. The estimated allele frequency in Tennessee Walking Horses is 10%. Main limitations Limited phenotype information for controls and no additional cases with which to replicate this finding. Conclusions We identified a likely causal recessive missense variant in GRM6. Based on protein modelling, this variant alters GRM6 binding, and thus signalling from the retinal rod cell to the ON-bipolar cell, impairing vision in low light conditions. Given the 10% population allele frequency, it is likely that additional affected horses exist in this breed and further work is needed to identify and examine these animals.

Cancer-associated fibroblasts (CAF) are important constituents of the tumor microenvironment (TME) and are major drivers of tumorigenesis. Yet, therapies aiming at eliminating CAF have failed to cure patients. This setback has raised questions regarding whether CAF exclusively favour cancer progression, or if they may also assume tumor-suppressor functions. In the present study, we used proteomics and single cell RNA-sequencing analysis to examine the CAF landscape in hepatocellular carcinoma (HCC). We thereby unveil three major CAF populations in HCC, one of which specifically expressing the prolargin protein. This CAF subpopulation (further termed as CAF_Port) shared a strong transcriptomic signature with portal liver fibroblasts. We further show that CAF_Port deposit prolargin in the TME and that its levels are lower in tumors as compared to the peritumoral region. Mechanistically, aggressive cancer cells degraded prolargin using matrix metalloprotease activity. Survival analysis of 188 patients revealed that high prolargin protein levels correlate with good patient outcome (HR = 0.37; p = 0.01). In vivo, co-injection of cancer cells with fibroblasts silenced for prolargin, led to faster tumor development (5-fold; p = 0.01), mainly due to stronger angiogenesis. Using protein-protein interaction study and structural modelling, we further demonstrate that prolargin binds and inhibits the activity of several pro-agiogenic proteins, including hepatocyte and fibroblast growth factors. In conclusion, prolargin is angiogenesis modulator and CAF-derived tumor suppressor in HCC. Stabilizing prolargin levels in the CAF_Port subpopulation may revert their tumor-antagonizing properties, warranting exploration in further pre-clinical studies.

Hepatocellular carcinoma (HCC) is a difficult to cure primary liver cancer. Recent breakthroughs in cancer treatment highlight the importance of the tumor microenvironment (TME) and its ability to promote or suppress tumor development. While understanding of this dual behaviour is expanding for immune cells, little is known for other stromal cells, and notably cancer-associated fibroblasts (CAF). Here, we unveil a novel CAF tumor-antagonizing protein, prolargin, and unravel its regulation and mechanism of action in human HCC. Employing proteomics and single cell RNA sequencing data analysis we investigated prolargin expression and the cell of origin in human HCC. The significance for clinical outcome was examined in a cohort of HCC patients (N=188), while prolargin function was studied in vivo using orthotopic models. Biochemical studies along with structural modelling/docking were employed to elucidate the mechanism of action. Our findings show that the expression of prolargin is confined to portal fibroblast-derived CAF. Prolargin is secreted in the TME where its quantity positively correlated with patient outcome (HR=0.37; p=0.01). Aggressive HCC cells reduce prolargin levels mainly through matrix metalloprotease 3 (MMP3) activity. In vivo, tumors with lower prolargin expression displayed faster progression (5-fold; p=0.01) and stronger angiogenesis. Models of prolargin structure revealed a solenoid (horseshoe) folding, favouring its binding and inhibition of several growth factors, except vascular endothelial growth factor (VEGF). MMP-inhibition (batimastat) combined with VEGFR targeting (sorafenib) in vivo demonstrated superior tumor control compared to sorafenib treatment alone. Prolargin-expressing fibroblasts have tumorantagonizing properties and stabilizing prolargin expression should be considered as therapeutic strategy for HCC.

The recombinant adeno-associated virus (rAAV) vector has been successfully employed in clinical trials for patients with blindness and bleeding diseases as well as neuromuscular disorders. To date, it remains a major challenge to achieve higher transduction efficiency with a lower dose of rAAV vector. Our previous studies have demonstrated that serum proteins are able to directly interact with AAV virions for transduction enhancement. Herein, we explored the effect of the FerA domains, which are derived from ferlin proteins and possess membrane-fusion activity, on AAV transduction. Our results show that FerA domains from dysferlin, myoferlin, and otoferlin proteins are able to directly interact with AAV vectors and enhance AAV transduction in vitro and in mice through either intravenous or intramuscular injections. The enhanced AAV transduction induced by human/mouse FerA domains is achieved in various cell lines and in mice regardless of AAV serotypes. Mechanism studies demonstrated that the FerA domains could effectively enhance the ability of AAV vectors to bind to target cells and cross the vascular barrier. Additionally, FerA domains slow down the blood clearance of AAV. Systemic administration of AAV8/hFIX-FerA complex induced approximate 4-fold more human coagulation factor IX expression and improved hemostasis in hemophilia B mice than that of AAV8/hFIX. Collectively, we show, for the first time, that multiple FerA domains could be tethered on the AAV capsid and enhance widespread tissue distribution in an AAV serotypes-independent manner. This approach therefore holds a promise for future clinical application.

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain ‘mini-myoferlin’. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate ‘mini-myoferlin’ can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

ATP-binding cassette proteins are ubiquitously present throughout all known genomes. Their basic functional unit possesses two transmembrane domains and two nucleotide-binding domains. The nucleotide-binding domains are responsible for ATP binding and hydrolysis, and their 3-dimensional structure is conserved across ATP-binding cassette proteins. Binding of ATP produces nucleotide-binding domain dimerization, a step necessary for hydrolysis. However, the possibility that nucleotide-binding domains bind and/or hydrolyze nucleotide triphosphates different from ATP has not been explored in detail. Here, we studied that possibility using M. jannaschii MJ0796, a prototypical ATP-binding cassette nucleotide-binding domain. We found that nucleotide-binding domain dimerization occurs as a result of binding to the natural nucleotide triphosphates ATP, GTP, CTP and UTP, and also to the analog ATP-γ-S. All the natural nucleotide triphosphates are hydrolyzed at similar rates, whereas ATP-γ-S is not hydrolyzed. We also found that the non-hydrolyzable ATP analog AMP-PNP, frequently assumed to produce the nucleotide-bound conformation, failed to elicit nucleotide-binding domain dimerization. Our results raise the possibility that not all the nucleotide binding sites of nucleotide-binding domains are occupied by ATP under physiological conditions, and that ATP is not always the nucleotide hydrolyzed to dissociate the nucleotide-binding domain dimers.

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-β reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aβ by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aβ assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

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.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.

Amyloids are traditionally considered pathological protein aggregates that play causative roles in neurodegenerative disease, diabetes and prionopathies. However, increasing evidence indicates that in many biological systems nonpathological amyloids are formed for functional purposes. In this review, we will specifically describe amyloids that carry out biological roles in sexual reproduction including the processes of gametogenesis, germline specification, sperm maturation and fertilization. Several of these functional amyloids are evolutionarily conserved across several taxa, including human, emphasizing the critical role amyloids perform in reproduction. Evidence will also be presented suggesting that, if altered, some functional amyloids may become pathological.

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.

Systemic Lupus Erythematosus (SLE) is a debilitating autoimmune disease with poly- and mono-genic etiologies. One cause of monogenic SLE is the disruption of Dnase1L3. Dnase1L3 is a serum endonuclease that degrades chromatin in apoptotic microparticles. Dnase1L3 could represent a novel therapy for some cases of SLE. However, the structure of Dnase1L3 is predicted by the 42% identical protein Dnase1. To better understand the structure-function of Dnase1L3, we set out to solve the crystal structure. To solve the crystal structure, we first generated a recombinant His6-Maltose Binding Protein-Dnase1L3 fusion protein. We developed a protocol for purifying Dnase1L3. We isolated Dnase1L3 from the cell lysate using Nickel-NTA beads in maltose buffer, removed purification tags on Dnase1L3 with Tobacco Etch Virus protease, and purified Dnase1L3 on a negatively charged ion exchange column. We then purified Dnase1L3 by size exclusion chromatography to 96.7% final purity. We verified purity at each step by Coomassie staining and, confirmed identity of the final product by Western blot. Recombinant Dnase1L3 robustly degraded plasmid DNA, with a specific activity of 1.83 x 104 U/mg. We found Dnase1L3 had a propensity for small mosaic crystals in a Tris and MgCl2 crystallization buffer. However, larger crystals were grown at 7 °C, compared to 22 °C. Overall, we have developed a new purification method for generating active Dnase1L3 useful for crystallography and other high-purity applications. These applications will further our understanding of Dnase1L3 structure and its function in SLE.

Glutaredoxin from the cyanobacterium Synechocystis sp. PCC 6803 is a small protein, containing only 88 amino acids, that participates in a large number of redox reactions, serving both as an electron donor for enzyme-catalyzed reductions and as a regulator of diverse metabolic pathways. The crystal structures of glutaredoxins from several species have been solved, including the glutaredoxin A isoform from the cyanobacterium Synechocystis sp. PCC 6803. We have utilized the small size of Synechocystis glutaredoxin A and its propensity to form protein crystals that diffract to high resolution to explore a long-standing question in biochemistry; i.e., what are the effects of mutations on protein structure and function? Taking advantage of these properties, we have initiated a long-term educational project that would examine the structural and biochemical changes in glutaredoxin as a function of single-point mutational replacements. Here, we report some of the mutational effects that we have observed to date.

Occludin is a tight-junction protein controlling the integrity of endothelial and epithelial cell layers. It forms complexes with the cytoplasmic proteins ZO-1, ZO-2 and ZO-3. The ZO-binding domain in the C-terminal cytoplasmic region of human occludin has previously been isolated and identified. This domain, as expressed in a bacterial system or isolated from native cellular occludin, maintains its ability to bind ZO-1 and ZO-2. The crystallization conditions of the human ZO-binding domain are reported here. The crystals diffract to 2.3 A resolution and were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 33.3, b = 35.4, c = 107.3 A.

Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers (excimers) by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene (pyrene-4-maleimide), with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes.