A method for probing the mutational landscape of amyloid structure

Motivation: Proteins of all kinds can self-assemble into highly ordered β-sheet aggregates known as amyloid fibrils, important both biologically and clinically. However, the specific molecular structure of a fibril can vary dramatically depending on sequence and environmental conditions, and mutations can drastically alter amyloid function and pathogenicity. Experimental structure determination has proven extremely difficult with only a handful of NMR-based models proposed, suggesting a need for computational methods. Results: We present AmyloidMutants, a statistical mechanics approach for de novo prediction and analysis of wild-type and mutant amyloid structures. Based on the premise of protein mutational landscapes, AmyloidMutants energetically quantifies the effects of sequence mutation on fibril conformation and stability. Tested on non-mutant, full-length amyloid structures with known chemical shift data, AmyloidMutants offers roughly 2-fold improvement in prediction accuracy over existing tools. Moreover, AmyloidMutants is the only method to predict complete super-secondary structures, enabling accurate discrimination of topologically dissimilar amyloid conformations that correspond to the same sequence locations. Applied to mutant prediction, AmyloidMutants identifies a global conformational switch between Aβ and its highly-toxic ‘Iowa’ mutant in agreement with a recent experimental model based on partial chemical shift data. Predictions on mutant, yeast-toxic strains of HET-s suggest similar alternate folds. When applied to HET-s and a HET-s mutant with core asparagines replaced by glutamines (both highly amyloidogenic chemically similar residues abundant in many amyloids), AmyloidMutants surprisingly predicts a greatly reduced capacity of the glutamine mutant to form amyloid. We confirm this finding by conducting mutagenesis experiments.National Institutes of Health (U.S.) (grant 1R01GM081871)National Institutes of Health (U.S.) (grant GM25874

Cytoskeleton and anchoring junctions in the testis: emerging concepts for the regulation of junctionintegrity

published_or_final_versionBiological SciencesDoctoralDoctor of Philosoph

Signalling pathways regulating the blood–testis barrier

Throughout mammalian spermatogenesis, preleptotene/leptotene spermatocytes traverse the blood–testis barrier during stages VIII–XI of the seminiferous epithelial cycle while trapped within a dynamic intermediate compartment that is sealed at north and south poles by tight junctions, basal ectoplasmic specializations, desmosomes and gap junctions. In order for spermatocytes to gain entry into the adluminal compartment of the seminiferous epithelium for continued development, \u27old\u27 junctions present above migrating spermatocytes disassemble, while \u27new\u27 junctions assemble simultaneously below these germ cells. In this way, the integrity of the blood–testis barrier and the homeostasis of the seminiferous epithelium can remain intact during spermatogenesis. Previous studies have shown an array of cellular events, including protein internalization and cytoskeletal remodeling, to underline blood-testis barrier restructuring, whereas other studies have reported BTB dysfunction to associate with activation of the p38 mitogen-activated protein kinase pathway. Herein, we discuss the signaling pathways and mechanisms involved in blood–testis barrier restructuring in the mammalian testis

The biology of interleukin-1: Emerging concepts in the regulation of the actin cytoskeleton and cell junction dynamics

Interleukin (IL)-1 is a proinflammatory cytokine with important roles in innate immunity, as well as in normal tissue homeostasis. Interestingly, recent studies have also shown IL-1 to function in the dynamics of the actin cytoskeleton and cell junctions. For example, treatment of different epithelia with IL-1α often results in the restructuring of the actin network and cell junctions, thereby leading to junction disassembly. In this review, we highlight new and interesting findings that show IL-1 to be a critical player of restructuring events in the seminiferous epithelium of the testis during spermatogenesis

Coordinating cellular events during spermatogenesis: A biochemical model

Throughout spermatogenesis, a select pool of germ cells, the leptotene spermatocytes, must traverse the blood-testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium. This event requires extensive restructuring of cell junctions, and it must also coincide with germ cell cycle progression in preparation for primary spermatocyte meiosis. Recent findings show that cell-cycle-associated kinases and phosphatases, including mitogen-activated protein kinases (MAPKs), participate in the pathways that also direct germ cell adhesion and movement. Our new biochemical model explains, in part, how two distinct cellular events, BTB restructuring and spermiation, are coordinated to maintain spermatogenesis and fertility. In this way, MAPKs would synchronize cell cycle progression in primary spermatocytes with junction remodeling and cell migration across the BTB

The biology of the desmosome-like junction: A versatile anchoring junction and signal transducer in the seminiferous epithelium

Mammalian spermatogenesis, a complex process that involves the movement of developing germ cells across the seminiferous epithelium, entails extensive restructuring of Sertoli-Sertoli and Sertoli-germ cell junctions. Presently, it is not entirely clear how zygotene spermatocytes gain entry into the adluminal compartment of the seminiferous epithelium, which is sealed off from the systemic circulation by the Sertoli cell component of the blood-testis barrier, without compromising barrier integrity. To begin to address this question, it is critical that we first have a good understanding of the biology and the regulation of different types of Sertoli-Sertoli and Sertoli-germ cell junctions in the testis. Supported by recent studies in the field, we discuss how crosstalk between different types of junctions contributes to their restructuring during germ cell movement across the blood-testis barrier. We place special emphasis on the emerging role of desmosome-like junctions as signal transducers during germ cell movement across the seminiferous epithelium

Dynamins, spermatogenesis and contraceptive development

Dynamins are large GTPases of ∼100 kDa known to participate in endocytosis and interact with the actin-based cytoskeletal network in multiple tissues. Recent studies have shown that dynamins play a critical role in the internalization of integral membrane proteins via either clathrin-mediated or clathrin-independent endocytosis. Furthermore, recent studies have shown that dynamin II interacts with junctional complex adaptors, namely ZO-1 and β-catenin, at the blood-testis barrier in the seminiferous epithelium of adult rat testes. This interaction may be responsible for pulling away tight junction- and adherens junction-based protein complexes, thereby facilitating blood-testis barrier opening to permit preleptotene and leptotene spermatocyte migration, which is a critical event in spermatogenesis occurring at stage VIII of the seminiferous epithefial cycle. In this short review, we highlight some of the latest findings on dynamins in the field, and discuss how this information can be used to further expand the functional studies to tackle the role of dynamins in spermatogenesis. It is likely that dynamins per se or their interacting protein partners can become a target for male contraceptive research to compromise spermatogenesis, leading to transient male infertility without perturbing the hypothalamic-pituitary-testicular axis

Interleukin-1α is a regulator of the blood-testis barrier

Throughout spermatogenesis, the Sertoli cell blood-testis barrier (BTB) is strictly regulated by cytokines, which mediate its timely restructuring, thereby allowing spermatocytes to enter the adluminal compartment of the seminiferous epithelium for development into spermatozoa. The aim herein was to investigate whether germ cells play a role in BTB restructuring via the action of interleukin-1α (IL-1α) since germ cells are known to control Sertoli cell production of this cytokine, and if yes, how these effects are mediated. When Sertoli cells were isolated from Sprague-Dawley rats and plated at high density, IL-1α (100 pg/ml) was shown to “open” the Sertoli cell barrier when its integrity was assessed by transepithelial electrical resistance measurements. Further investigation of Sertoli cells treated with IL-1α revealed striking changes in the cellular distribution of actin filaments when compared to untreated cells. These effects at the Sertoli cell barrier were mediated, in part, by epidermal growth factor receptor pathway substrate 8 (Eps8; an actin bundling and barbed-end capping protein) and actin-related protein 3 (Arp3; a component of the actin nucleation machinery). As important, an increase in the kinetics of occludin internalization but a decrease in its rate of degradation was noted following IL-1α treatment. These results indicate that IL-1α is a critical regulator of BTB dynamics.—Lie, P. P. Y., Cheng, C. Y., Mruk, D. D. Interleukin-1α is a regulator of the blood-testis barrier

Focal adhesion kinase and actin regulatory/binding proteins that modulate F-actin organization at the tissue barrier: Lesson from the testis

Focal adhesion kinase (FAK), as its name implied, is an important mediator of integrin-based signaling function in mammalian cells at the focal adhesion complex (FAC, also known as focal contact) at the cell-extracellular matrix interface. FAK is intimately related to cell movement, such as in macrophages, fibroblasts and also tumor cells. In the testis, however, FAK and two of its phosphorylated forms, p-FAK-Tyr^407 and -Tyr^397, are not found at the FAC since there is no ultrastructure analogous or similar to FAC in the mammalian testis vs. other epithelia. Instead, FAK and its two phosphorylated forms are detected along the seminiferous epithelium in the rat testis at the cell-cell interface in a testis-specific adherens junction (AJ) known as the ectoplasmic specialization (ES). ES is an F-actin-rich ultrastructure in which bundles of actin filaments are sandwiched in-between plasma membrane and cisternae of endoplasmic reticulum not found in other mammalian epithelial/endothelial cells. The ES is restricted to the interface of Sertoli cells and spermatids (step 8–19) known as the apical ES, and to the Sertoli cell-cell interface known as the basal ES. Interestingly, the basal ES is also an integrated component of the blood-testis barrier (BTB), coexisting with tight junction (TJ) and gap junction (GJ), and it is conceivable that actin filament bundles at the ES undergo extensive organization, converting from their “bundled” to “de-bundled/branching” configuration to facilitate transport of germ cells across the epithelium and at the BTB during the epithelial cycle. A recent report (Lie et al. PNAS 109:12562–12567, 2012) has demonstrated that the stage-specific and spatiotemporal expression of p-FAK-Tyr^407 and -Tyr^397 are crucial to the regulation of these events via their stage-specific and spatiotemporal expression during the epithelial cycle mediated by their effects on the organization of the actin filament bundles at the ES, involving actin binding/regulatory proteins. In this Commentary, we will critically evaluate these findings in light of other recent reports in the field. While these ideas are based on studies in the BTB in the rat testis, this information should be applicable and helpful to investigators studying other tissue barriers