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Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.

Uncoordinated 119 protein (UNC119) is a partner of transducin-α subunit (Gαt ) that is essential for transducin trafficking in rod photoreceptors. The interaction is known to involve binding of the acylated N terminus of Gαt to the hydrophobic pocket of UNC119. To gain insights into the mechanism of transducin trafficking, we isolated a highly pure protein complex between myristoylated chimeric Gαt (Gαt *) and UNC119₅₀₋₂₄₀, and examined the solution structure by small angle X-ray scattering and chemical crosslinking. The solution structure of the Gαt -UNC119₅₀₋₂₄₀ complex was derived with rigid body/ab initio modeling against the small angle X-ray scattering data by use of known atomic structures of Gαt and UNC119, and a distance constraint based on the protein crosslinking with p-phenyldimaleimide. The model of the Gαt -UNC119₅₀₋₂₄₀ complex indicates rotation and bending of the N-terminal α-helix of Gαt from its position in the structure of the heterotrimeric G-protein transducin (Gt ). This allows a considerably more compact complex conformation, which also suggests a novel interface involving the switch II/α3-β5 surface of Gαt . Supporting a novel interface, UNC119 was found to bind full-length Gαt * more strongly than the Gαt N-terminal peptide. Furthermore, UNC119 competed with the effector molecule phosphodiesterase-6 γ-subunit, which is known to bind to the same surface of Gαt . The solution structure of the Gαt -UNC119 complex suggests that the ability of UNC119 to dissociate Gt subunits and release Gαt from the membrane is attributable to disruption and sterical occlusion of the Gβ₁γ₁-binding sites on Gαt .

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

Gold nanoparticles (Au NPs, 20 nm) were conjugated with two different cysteine-terminated peptides. Radio-ligand binding studies were conducted to characterize Au NP-peptide binding, suggesting both covalent and noncovalent interactions. The interactions of serum proteins with Au NP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugates (15 minutes). The cellular uptake of free peptides and nanoconjugates into mouse myogenic (Sol8) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence signals compared to those in the presence of serum (P < 0.05), suggesting that serum proteins inhibit Au NP-mediated peptide delivery. The cellular uptake of nanoconjugates was also confirmed using transmission electron microscopy. These data suggest that Au NP-peptide nanoconjugates are a useful platform for intracellular delivery of therapeutics. However, a deeper understanding of the mechanisms regulating their uptake and intracellular trafficking is needed.

Adipocyte differentiation (AD) and AD-specific gene expression was studied in 3T3-L1 cells in response to oleic acid (OA) or linoleic acid (LA) alone and in combination with insulin. This system facilitated the study of key regulators of adipogenesis PPARγ and C/EBPα and other AD-specific genes, in the absence of dexamethasone (DEX) and isobutyl-1-methyl xanthine (IBMX) (components of the traditional AD medium, DMI). Lipid accumulation and expression levels of AD-specific genes were enhanced by both OA and LA in the presence of insulin but not by OA or LA alone. Gene expression levels of PPARγ, C/EBPα, FABP4, and SREBP1c induced by OA plus insulin, were comparable to DMI medium, by study day 10. The response to long-chain fatty acids (LCFA) plus insulin in the presence or absence of LY294002 demonstrated that the insulin-induced PI 3-kinase pathway regulates AD and AD-specific gene expression levels. Insulin treatment in the presence or absence of genistein suggested that genistein invoked inhibition of AD and AD-specific gene expression. In contrast when LCFA were also included with insulin, the presence of genistein invoked a pronounced and opposite effect on AD to that in the absence of LCFA. This effect may be modulated via C/EBPα as C/EBPα but not PPARγ expression patterns closely reflected the changes in AD. DMI invoked a rapid expression of all genes studied, and LCFA plus insulin invoke more gradual increases in gene expression, to similar levels to those invoked by DMI. The model system is valuable for study of transactivators and response elements of PPARγ and C/EBPα genes.

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.

Photoreceptor phosphodiesterase-6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS remains largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing cGMP-binding, cGMP-specific phosphodiesterase-5/PDE6 chimeric proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal. Trafficking of phosphodiesterase 6 (PDE6), the key phototransduction enzyme, was analyzed in rods of transgenic X. laevis by constructing transgenes for rod-specific expression of chimeric PDE6/PDE5 proteins. Analysis of localization of chimeric PDE proteins reveals a novel GAFa domain–dependent targeting signal involved in proper trafficking of PDE6 to the rod outer segment.

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2'-N-(pyren-1-yl)carbonyl-2'-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (Φ(F) = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

Skeletal muscle regeneration following damage is mainly attributed to activation, proliferation and myogenic differentiation of muscle stem cells known as satellite cells. Satellite cells lie beneath the basal lamina that surrounds muscle fibres. These pluripotent cells respond to muscle injury to initiate the process of satellite cell myogenesis, regulated by several transcription factors known as myogenic regulatory factors (MRFs): MyoD, Myf5, myogenin and MRF4. Pax-7-expressing quiescent satellite cells, upon activation, up-regulate MyoD and Myf5 expression to generate myogenic lineage cells, or myoblasts. Myoblasts proliferate and subsequently up-regulate myogenin and MRF4 levels leading to terminal differentiation into muscle fibres (Charge & Rudnicki, 2004). On the other hand, insulin-like growth factor−1 (IGF-1) signalling was implicated in the process of myogenesis. In vitro studies demonstrated that IGF-1 positively regulated myogenesis in a two-step process, first by cell proliferation of stem cells and later by enhancing differentiation process (Rosenthal & Cheng, 1995; Engert et al. 1996). Transgenic mice with muscle-specific IGF-1 isoform supplementation have muscle regenerative capacity in advanced ages, suggesting muscle-specific IGF-1 in the regenerative process (Musaro et al. 2001). To date, three isoforms of IGF-1 have been detected in skeletal muscle: IGF-1Ea, IGF-1Eb and IGF-1Ec (IGF-1Ec is referred to as mechano-growth factor, MGF) (Rotwein, 1986; Yang et al. 1996). The roles of these isoforms in muscle repair, particularly their regulation of MRFs, was not elucidated in detail. A recent report by McKay et al. (2008) in The Journal of Physiology investigated the temporal gene expression of MRFs and IGF-1 isoforms in human quadriceps femoris upon exercise-induced muscle damage. Eight male subjects were recruited to perform intensive muscle-lengthening exercise. Muscle biopsies were collected before (0 h), and after exercise at 4, 24, 72 and 120 h. Immunofluorescence assays were done to determine localization of IGF-1 and Pax-7 proteins after the exercise-induced damage. The authors speculate about the interaction of IGF-1 signalling and transcription of MRFs based on gene expression and immunoflourescence data. In summary, MyoD and Myf5 gene expressions were significantly up-regulated at 4 h and 24 h post-exercise, respectively. Up-regulation of myogenin gene expression was observed from 4 h onwards, and MRF4 expression was significantly up-regulated at 72 and 120 h. These observations are partly inconsistent with accepted cascade of myogenic differentiation, as the authors observed up-regulation of myogenin prior to Myf5. This clearly suggests limitations of interpretations based on crude muscle samples and gene expression analyses to acutely delineate a cascade in response to damage. The authors also measured transcript levels of IGF-1Ea, IGF-1Eb and MGF. MGF and IGF-1Ea were significantly up-regulated at 24 h and 72 h, respectively, whereas IGF-1Eb was up-regulated at 72 and 120 h. These data were then used to determine correlations between IGF-1 isoforms and MRF gene expressions. The authors report a correlation of MGF with Myf5, while IGF-1Ea and IGF-1Eb correlated with MRF4 expression. The observed correlations are limited in their capacity to provide biological information, because IGF-1Ea and IGF-1Eb correlated with only one of the differentiation markers (MRF4), and MGF was correlated with only one of the myogenic precursor markers (Myf5). Adding to this conundrum as noted above is the paradoxical up-regulation of myogenin before Myf5. In essence, the gene expression analyses along with statistical analyses are limited to provide insight into signalling pathways connecting IGF-1 isoforms and MRFs. The observations from similar studies were contradictory to the current report; for example, Psilander et al. (2003) reported that MyoD was up-regulated immediately after exercise and reached baseline after 1 h onwards, whereas MRF4 and myogenin were significantly up-regulated only at 2 h and 6 h, respectively. It has to be noted that the changes in gene expression reported by Psilander et al. were rapid i.e. within 6 h of exercise. At later time-points the levels were not different from baseline. They report data suggesting a decline in IGF-1a levels at 1 h and 6 h post-exercise. These gene expression patterns are difficult to interpret and the apparent decline of IGF-1a in the muscle regeneration process is unclear. These apparent discrepancies in the timeline of gene expression changes and relationship of IGF-1 and MRFs may be attributed to limitations of interpretations solely based upon gene expression changes, different muscle injury protocols and biological variation in subjects chosen for the two studies. Interpretations based on gene expression analyses are highly influenced by temporal effects depending on kinetics of promoter activation (pulsatile association of transcription factors on promoter), pre-mRNA to mature mRNA formation and half-life of mature mRNA. Due to intrinsic differences in these regulatory processes among transcripts, several minutes to days may be necessary to reliably quantify gene expression changes in response to injury. Thus, interpretations supported by associated protein changes will provide strength to delineate muscle regenerative process. On the other hand, McKay et al. claim that IGF-1 protein was localized only in satellite cells at 24 h, but present both in satellite cells and myofibres at 72 h and 120 h post-exercise. Immunofluorescence data were difficult to interpret due to colocalization of DAPI with IGF-1 but, nonetheless, suggest that IGF-1 is localized to the nucleus of satellite cells. This is unexpected of IGF-1, a secreted protein that is localized in secretory vesicles. Thus, it is difficult to decipher biological information that links different isoforms of IGF and MRFs based on the immunoflourescence data. Although in vitro/rodent models laid foundations for fundamental understanding of muscle regeneration process, understanding details of myogenic regulatory mechanisms in human beings is needed for realization of potential for therapeutic interventions. In this regard, use of human subjects by authors is highly appreciated, as it has the potential for discovering physiologically relevant molecular mechanisms. Future studies should include an examination as to whether the regenerative process is influenced by the extent of muscle damage and associated inflammatory response upon injury. This can be accomplished by determining the levels of serum creatine kinase, an indicator of muscle damage, and also by quantifying the inflammatory response by histological measurement of invading neutrophils and macrophages in injured muscle. This could then be followed by further analyses to determine the relationship between serum creatine kinase, inflammatory response and expression profiles of MRFs/IGF-1 isoforms. The extent of the regenerative process may also depend on basal satellite cell population, their activation and proliferation. In this context, measuring transcript levels of satellite cell marker Pax-7 will provide in indirect measure of relative satellite cell population/activity at different stages of the repair process. The presence of three isoforms of IGF-1 in muscle and their putative roles in repair underlines the importance of understanding their complicated regulatory and signalling mechanisms. Toward understanding IGF-1 biology in the muscle regenerative process, it is important to understand if one or more of the MRFs directly/indirectly regulate transcription or splicing of IGF-1 genes. There is a greater need for distinguishing the three isoforms of IGF-1 at the protein level and quantifying each of these isoforms, and also to determine their signalling. This becomes complex, as IGF proteins upon secretion bind to IGF-binding proteins (IGFBPs). There are several IGFBPs, and specific interactions of the IGF isoforms and IGFBPs may lead to different results, such as inhibitition or stimulation of IGF-1 signalling. This complexity can be partially addressed by conducting studies using transgenic rodent models with over-expression of each of the splice variants in muscle and determine IGF signalling and the myogenic process after injury. Further research in vitro may also be used to determine isoform-specific IGF signalling to gene expression of different MRFs, which involves determining the activated transcription factors and their responsive cis-elements on different MRFs. In addition, recent genetic models such as zebrafish can also be used to better understand the fundamentals of molecular regulation of the myogenic process, as the mechanisms of embryonic muscle development are similar to muscle regeneration. In conclusion, there is a clear need for complementary approaches using different models to understand regulatory and signalling mechanisms of each of the IGFs and MRFs. In the long term, studies using these approaches will help us to better understand and treat muscle degeneration.

Obesity contributes to the development of insulin resistance, hyperglycaemia and metabolic syndrome (Kahn & Flier, 2000; Kahn et al. 2006). However, effects of obesity on the control of skeletal muscle mass and hypertrophy are not well understood. Muscle hypertrophy is marked by an increase in protein synthesis and addition of contractile filaments to generate muscle force (Glass, 2003). A vast array of signalling networks in response to external stimuli including insulin, insulin growth factor-1 (IGF-1), steroid hormones and also amino acids mediate hypertrophy of skeletal muscle (Glass, 2003). Though the external signals such as insulin and IGF-1 activate their specific signalling, they share common downstream signalling molecules such as PI3-K and Akt. IGF-1/insulin act on their cell surface specific receptors and initiate respective signalling by activation of PI3K followed downstream signalling via activation of Akt (Cheatham & Kahn, 1995; Stitt et al. 2004). The mechanism of distinct actions of these peptide hormones despite activating common signalling molecule: Akt is beginning to be understood. Reported studies suggest that different isoforms of Akt (Akt1, Akt2 and Akt3) confer signal specificity of insulin in glucose metabolism and IGF-1 in growth. Mutational analyses revealed that Akt1-deficient mice have retarded growth and perinatal mortality, where as Akt2 knockout mice have no growth defects, but have impaired glucose metabolism (Chen et al. 2001; Cho et al. 2001a,b;). On the other hand, Akt3 knockout mice had normal body weights and glucose metabolism, but reduced brain sizes (Garofalo et al. 2003). In general, IGF-1 and also insulin to a lesser extent mediate protein synthesis by activating Akt1. The activated Akt1 increases protein synthesis by activating mammalian target of rapamycin (mTOR). mTOR increases protein synthesis by phosphorylating S6 protein kinase (p-S6K), a positive regulator of protein synthesis. mTOR also inhibits the activity of eIF4E binding protein-1 (4E-BP1), a negative regulator of the protein initiation factor, eukaryotic translation initiation factor 4E (eIF-4E) (Proud & Denton, 1997; von Manteuffel et al. 1997). Glycogen synthase kinase 3β (GSK3β) is another substrate of Akt, whose repression is known to induce hypertrophy, and its activity is regulated negatively by the phosphorylation of serine 9 (p-GSK-3β) (Rommel et al. 2001). In a recent issue of The Journal of Physiology, Sitnick et al. (2009) investigated load-induced hypertrophy and underlying molecular mechanisms in response to high-fat diet. The authors investigated whether a high-fat diet (HFD) would alter the ability of skeletal muscle to respond to functional overload (FO). Further, they determined if underlying signalling mechanisms of hypertrophy are compromised in mice fed a high-fat diet. As expected, mice increased their body size in response to HFD. Measurements of fat (epididymal and retroperitoneal fat pads) and muscle (gastrocnemius and soleus) suggested that increases in body size were primarily due to increased fat composition in the body. The authors also determined that mice fed a high-fat diet were hyperinsulinemic, suggesting development of insulin resistance potentially mediated by increased intra-myocellular triglycerides in muscle. On all the tested days there was a significant increase in the mass of pantaris muscle in response to FO mice compared to control. After 14 and 30 days of FO the authors found a 10 and 16% reduction in the growth of the plantaris muscle in the HFD versus the low-fat diet (LFD) mice, suggesting a moderate decrease in the growth of plantaris muscle in response to HFD. The authors noticed that extended HFD nutrition (30 weeks) had a dramatic effect, showing no difference in the mass of plantarius muscle compared to control mice. These observations suggest that a HFD diet has a negative effect on the load-induced hypertrophy. The authors give interesting directions for the research into whether obesity may be a negative factor for load-induced muscular hypertrophy. On a path leading toward understanding the mechanism underlying this apparent relationship, the authors measured general protein translation and also status of the cell signalling molecules such as Akt, S6K1 and GSK3-β. The association of the ribosomal 40S and 60S subunits resulting in a 80S peak is an indicator of active translation process. The analyses demonstrate that the 80S peak in LFD mice was larger than in HFD mice suggesting that the translation process is greater in LFD mice compared to HFD. The authors further suggest that the activity of signalling molecules such as Akt and S6K1 were attenuated, but not for GSK3 β in FO/HFD mice compared FO/LFD mice. Though the analyses provided in the Sitnick et al. paper provide useful information about the status of the signalling molecules in hypertrophy, the data/analyses are not sufficient to provide accurate mechanistic insights. The potential link between obesity and hypertrophy at a mechanistic level may be delineated by further analyses of other important targets including IGF-1 expression and also using appropriate controls in the analyses. For example, the authors observed increased Akt phosphorylation in HFD control mice with apparent insulin resistance. This may be due to higher load on the plantaris muscle in the case of HFD mice due to an increased body mass. The measurement of IGF-1 levels would have been useful for interpreting information. Upon examining the Western blots of Akt presented in the paper, it is clear that total Akt levels increase in response to FO, irrespective of diet; however, the authors did not analyse if the total Akt levels show differences in response to FO in the two diets (LFD vs. HFD). The analyses are presented as a ratio of Akt Ser-373 to total Akt to indicate activity levels of Akt; but these analyses in the context of robust changes in total Akt levels will only provide limited information regarding Akt activity. The analyses would have been improved by probing for a housing-keeping protein such as GAPDH, and normalizing total Akt levels and also phosphorylated Akt levels to GAPDH and making comparisons between diets. The same suggestion applies to the quantification of the expression of S6K1, p-S6K1, GSK-3β and p-GSK-3β. All the molecules chosen in the study are not unique to either insulin or IGF-1, but common to both. It is not clear whether the HFD mice exhibited compromised hypertrophy due to attenuation of insulin signalling or IGF-1 signalling. In this context, future studies directed toward deciphering the specific proximal pathways such as IGF-1 serum levels/muscle expression of IGF-1, IGF-1/insulin receptor activation and also further study of specific Akt isoforms may begin to provide a molecular basis for the apparent relationship between obesity and hypertrophy. On the other hand, it has to be determined if obesity decreases activity of the mice. In summary, the recent paper by Stinick et al. suggests that diet plays a role in the hypertrophy of skeletal muscle, although the exact mechanisms for this process remain unclear. The authors provide a base upon which further investigations can build to elucidate the complex signalling mechanisms of diet and their influence on load-induced hypertrophy.