KISS1R and ANKRD31 Cooperate to Enhance Leydig Cell Gene Expression via the Cytoskeletal-Nucleoskeletal Pathway.

Kisspeptins are involved in the regulation of hypothalamic-pituitary-gonadal axis, Leydig cell functions, and testosterone secretion, acting as endogenous ligands of the KISS1 receptor. ANKRD31 protein participates in male fertility, regulating meiotic progression, and epididymal sperm maturation. Here, we show that in Leydig cells, KISS1 receptor and ANKRD31 proteins physically interact; the formation of this protein complex is enhanced by Kisspeptin-10 that also modulates F-actin synthesis, favoring histone acetylation in chromatin and gene expression via the cytoskeletal-nucleoskeletal pathway. Kp/KISS1R system deregulation, expression impairment of cytoskeletal-nucleoskeletal mediators, Leydig gene targets, and the decreased testosterone secretion in Ankrd31 -/- testis strongly supported our hypothesis. Furthermore, cytochalasin D treatment subverted the gene expression induction dependent on Kisspeptin-10 action. In conclusion, the current work highlights a novel role for the Kisspeptin-10 in the induction of the cytoskeletal-nucleoskeletal route, downstream a physical interaction between KISS1 receptor and ANKRD31, with gene expression activation as final effect, in Leydig cells.

Sequence and Structure-Based Analyses of Human Ankyrin Repeats.

Ankyrin is one of the most abundant protein repeat families found across all forms of life. It is found in a variety of multi-domain and single domain proteins in humans with diverse number of repeating units. They are observed to occur in several functionally diverse proteins, such as transcriptional initiators, cell cycle regulators, cytoskeletal organizers, ion transporters, signal transducers, developmental regulators, and toxins, and, consequently, defects in ankyrin repeat proteins have been associated with a number of human diseases. In this study, we have classified the human ankyrin proteins into clusters based on the sequence similarity in their ankyrin repeat domains. We analyzed the amino acid compositional bias and consensus ankyrin motif sequence of the clusters to understand the diversity of the human ankyrin proteins. We carried out network-based structural analysis of human ankyrin proteins across different clusters and showed the association of conserved residues with topologically important residues identified by network centrality measures. The analysis of conserved and structurally important residues helps in understanding their role in structural stability and function of these proteins. In this paper, we also discuss the significance of these conserved residues in disease association across the human ankyrin protein clusters.

Ankrd31 in Sperm and Epididymal Integrity.

Ankyrin proteins (ANKRD) are key mediators linking membrane and sub-membranous cytoskeletal proteins. Recent findings have highlighted a new role of ANKRD31 during spermatogenesis, elucidating its involvement in meiotic recombination and male germ cell progression. Following testicular differentiation, spermatozoa (SPZ) enter into the epididymis, where they undergo several biochemical and enzymatic changes. The epididymal epithelium is characterized by cell-to-cell junctions that are able to form the blood-epididymal barrier (BEB). This intricate epithelial structure provides the optimal microenvironment needed for epididymal sperm maturation. To date, no notions have been reported regarding a putative role of ANKRD31 in correct BEB formation. In our work, we generated an Ankrd31 knockout male mouse model (Ankrd31-/- ) and characterized its reproductive phenotype. Ankrd31-/- mice were infertile and exhibited oligo-astheno-teratozoospermia (a low number of immotile SPZ with abnormal morphological features). In addition, a complete deregulation of BEB was found in Ankrd31-/- , due to cell-to-cell junction anomalies. In order to suggest that BEB deregulation may depend on Ankrd31 gene deletion, we showed the physical interaction among ANKRD31 and some epithelial junction proteins in wild-type (WT) epididymides. In conclusion, the current work shows a key role of ANKRD31 in the control of germ cell progression as well as sperm and epididymal integrity.

Ecological Genomics and Evolution of Trichoderma reesei.

The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) is an efficient industrial cell factory for the production of cellulolytic enzymes used for biofuel and other applications. Therefore, researches addressing T. reesei are relatively advanced compared to other Trichoderma spp. because of the significant bulk of available knowledge, multiple genomic data, and gene manipulation techniques. However, the established role of T. reesei in industry has resulted in a frequently biased understanding of the biology of this fungus. Thus, the recent studies unexpectedly show that the superior cellulolytic activity of T. reesei and other Trichoderma species evolved due to multiple lateral gene transfer events, while the innate ability to parasitize other fungi (mycoparasitism) was maintained in the genus, including T. reesei. In this chapter, we will follow the concept of ecological genomics and describe the ecology, distribution, and evolution of T. reesei, as well as critically discuss several common misconceptions that originate from the success of this species in applied sciences and industry.

Protein Phosphatase 2A Regulates Cardiac Na+ Channels.

RATIONALE: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. OBJECTIVE: To define phosphatase pathways that regulate INa,L in vivo. METHODS AND RESULTS: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. CONCLUSIONS: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.

Structural basis for extremely strong binding affinity of giant ankyrins to LC3/GABARAP and its application in the inhibition of autophagy.

The Atg8/LC3/GABARAP family of proteins binds its physiological binding partners, which function in macroautophagy (hereafter autophagy), via recognition of their short linear motif, also known as the LC3-interactiong region (LIR) or Atg8-interacting motif (AIM). The AIM/LIR motif, with the consensus sequence [W/F/Y]xx[L/I/V], utilizes the aromatic and hydrophobic residues that bind on the surface of Atg8/LC3/GABARAP. Despite modest binding affinity, this interaction is essential for efficient autophagy. Here we highlight the recent paper by Li and collaborators who discovered the structural basis for a much stronger interaction between the LIR motif-containing peptides and LC3/GABARAP. Moreover, they showed that these peptides are potent and selective inhibitors of autophagy in cultured cells and in C. elegans.

Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Tetrodotoxin-resistant voltage-gated sodium channel Nav 1.8 constitutively interacts with ankyrin G.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel Nav 1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Nav 1.8 occurs in neurons of the CNS. In CNS neurons, Nav 1.2 and Nav 1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Nav 1.8 and ankG. GST pull-down assay and surface plasmon resonance technology revealed that Nav 1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Nav 1.2. An ion channel bearing the ankyrin-binding motif of Nav 1.8 displaced the endogenous Nav 1 accumulation at the axon initial segment of hippocampal neurons. Finally, Nav 1.8 and ankG co-localized in skin nerves fibers. Altogether, these results indicate that Nav 1.8 carries all the information required for its localization at ankG micro-domains. The constitutive binding of Nav 1.8 with ankG could contribute to the pathological aspects of illnesses where Nav 1.8 is ectopically expressed in CNS neurons.

EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology.

RATIONALE: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. OBJECTIVE: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. METHODS AND RESULTS: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. CONCLUSIONS: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.