Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF.

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.

Titin-actin interaction: PEVK-actin-based viscosity in a large animal.

Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5-1.0) than mice (N2BA:N2B ratio ~0.2). To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL) to SL of 2.15 µm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1-400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 µm and by 50% at 2.25 µm, suggesting a SL-dependent nature of viscosity that might prevent SL "overshoot" at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

Skeletal Muscle Anti-Atrophic Effects of Leucine Involve Myostatin Inhibition.

Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by real-time PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.

Expression of MuRF1 or MuRF2 is essential for the induction of skeletal muscle atrophy and dysfunction in a murine pulmonary hypertension model.

BACKGROUND: Pulmonary hypertension leads to right ventricular heart failure and ultimately to cardiac cachexia. Cardiac cachexia induces skeletal muscles atrophy and contractile dysfunction. MAFbx and MuRF1 are two key proteins that have been implicated in chronic muscle atrophy of several wasting states. METHODS: Monocrotaline (MCT) was injected over eight weeks into mice to establish pulmonary hypertension as a murine model for cardiac cachexia. The effects on skeletal muscle atrophy, myofiber force, and selected muscle proteins were evaluated in wild-type (WT), MuRF1, and MuRF2-KO mice by determining muscle weights, in vitro muscle force and enzyme activities in soleus and tibialis anterior (TA) muscle. RESULTS: In WT, MCT treatment induced wasting of soleus and TA mass, loss of myofiber force, and depletion of citrate synthase (CS), creatine kinase (CK), and malate dehydrogenase (MDH) (all key metabolic enzymes). This suggests that the murine MCT model is useful to mimic peripheral myopathies as found in human cardiac cachexia. In MuRF1 and MuRF2-KO mice, soleus and TA muscles were protected from atrophy, contractile dysfunction, while metabolic enzymes were not lowered in MuRF1 or MuRF2-KO mice. Furthermore, MuRF2 expression was lower in MuRF1KO mice when compared to C57BL/6 mice. CONCLUSIONS: In addition to MuRF1, inactivation of MuRF2 also provides a potent protection from peripheral myopathy in cardiac cachexia. The protection of metabolic enzymes in both MuRF1KO and MuRF2KO mice as well as the dependence of MuRF2 expression on MuRF1 suggests intimate relationships between MuRF1 and MuRF2 during muscle atrophy signaling.

Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

Small-molecule-mediated chemical knock-down of MuRF1/MuRF2 and attenuation of diaphragm dysfunction in chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small-molecule inhibitor of MuRF1 in treating CHF-induced diaphragm myopathy and loss of contractile function. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham-operated animals (sham) served as controls. One week post-left anterior descending coronary artery ligation animals were randomized into two groups-one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946-a recently described MuRF1-interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed. RESULTS: Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up-regulation of the MuRF1 and MuRF2 was attenuated after infarct. CONCLUSIONS: Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.

Small-molecule inhibition of MuRF1 attenuates skeletal muscle atrophy and dysfunction in cardiac cachexia.

BACKGROUND: Muscle ring finger 1 (MuRF1) is a muscle-specific ubiquitin E3 ligase activated during clinical conditions associated with skeletal muscle wasting. Yet, there remains a paucity of therapeutic interventions that directly inhibit MuRF1 function, particularly in vivo. The current study, therefore, developed a novel compound targeting the central coiled coil domain of MuRF1 to inhibit muscle wasting in cardiac cachexia. METHODS: We identified small molecules that interfere with the MuRF1-titin interaction from a 130 000 compound screen based on Alpha Technology. A subset of nine prioritized compounds were synthesized and administrated during conditions of muscle wasting, that is, to C2C12 muscle cells treated with dexamethasone and to mice treated with monocrotaline to induce cardiac cachexia. RESULTS: The nine selected compounds inhibited MuRF1-titin complexation with IC50 values <25 µM, of which three were found to also inhibit MuRF1 E3 ligase activity, with one further showing low toxicity on cultured myotubes. This last compound, EMBL chemical core ID#704946, also prevented atrophy in myotubes induced by dexamethasone and attenuated fibre atrophy and contractile dysfunction in mice during cardiac cachexia. Proteomic and western blot analyses showed that stress pathways were attenuated by ID#704946 treatment, including down-regulation of MuRF1 and normalization of proteins associated with apoptosis (BAX) and protein synthesis (elF2B-delta). Furthermore, actin ubiquitinylation and proteasome activity was attenuated. CONCLUSIONS: We identified a novel compound directed to MuRF1's central myofibrillar protein recognition domain. This compound attenuated in vivo muscle wasting and contractile dysfunction in cardiac cachexia by protecting de novo protein synthesis and by down-regulating apoptosis and ubiquitin-proteasome-dependent proteolysis.

Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.

Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors.

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63(-/-) mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.

Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1.

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

Regulation of nicotinic acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/lysosomal and atrophy pathways.

Muscle atrophy is a process of muscle wasting induced under a series of catabolic stress conditions, such as denervation, disuse, cancer cachexia, heart and renal failure, AIDS, and aging. Neuromuscular junctions (NMJs), the synapses between motor neurons and muscle fibers undergo major changes in atrophying muscles, ranging from mild morphological alterations to complete disintegration. In this study, we hypothesized that remodeling of NMJs and muscle atrophy could be linked together. To test this, we examined if a major atrophy-promoting E3 ubiquitin ligase, MuRF1, is involved in the maintenance of NMJs. Immunofluorescence revealed that MuRF1 is highly enriched close to the NMJ. Affinity precipitation and in vivo imaging showed that MuRF1 interacts in endocytic structures with both, acetylcholine receptor, the primary postsynaptic protein of the NMJ, as well as with Bif-1, an autophagy- and endocytosis-regulating factor. In vivo imaging, radio labeling, and weighing approaches demonstrated that metabolic destabilization of acetylcholine receptors and muscle atrophy induced by denervation were significantly rescued in MuRF1-KO animals. Notably, interaction with Bif-1, and the rescue of AChR lifetime and muscle atrophy were specific to MuRF1 but not MuRF2. Our data demonstrate an involvement of MuRF1 in membrane protein-turnover, including the degradation of AChRs at the NMJ under atrophying conditions where MuRF1 also interacts and associates with Bif-1.

MuRF1-dependent regulation of systemic carbohydrate metabolism as revealed from transgenic mouse studies.

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase beta (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.

Induction of MuRF1 is essential for TNF-alpha-induced loss of muscle function in mice.

BACKGROUND: Humoral circulating inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can impair skeletal muscle contractility. Furthermore, TNF-alpha expression correlates with elevated levels of atrogin-like muscle-specific ubiquitin E3 ligases, which are presumed to mediate muscle protein breakdown and atrophy. However, the casual relationships between MuRF1 and TNF-alpha and their relative contributions to muscle function impairment are not known. METHODS: TNF-alpha or saline was injected into either C57Bl6 or MuRF1(-/-) mice. After 16-24 h, the expression of MuRF1 in skeletal muscle was quantified by quantitative reverse transcription-PCR and Western blot analysis. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, two-dimensional gel electrophoresis was performed. RESULTS: Wild-type animals injected with TNF-alpha had higher MuRF1 mRNA expression (saline versus TNF-alpha: 56.6+/-12.1 versus 133.6+/-30.3 arbitrary units; p<0.05) and protein expression (saline versus TNF-alpha: 0.38+/-0.11 versus 1.07+/-0.25 arbitrary units; p<0.05) as compared to saline-injected littermates. Furthermore, TNF-alpha reduced force development at 150 Hz by 25% in C57Bl6 animals (saline versus TNF-alpha: 2412+/-120 versus 1799+/-114 g/cm(2); p<0.05), but not in MuRF1(-/-) mice (saline versus TNF-alpha: 2424+/-198 versus 2431+/-180 g/cm(2); p=NS). Proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T in wild-type animals treated with TNF-alpha as compared to MuRF1(-/-) mice that received TNF-alpha. CONCLUSION: The results of this study demonstrate for the first time that TNF-alpha-induced reduction in skeletal muscle force development depends on the induction of the atrophy-related E3 ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-alpha/MuRF1-mediated down-regulation of fast skeletal muscle troponin T.

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis.

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.

Structural and biochemical characterization of the yeast exosome component Rrp40.

The exosome is a protein complex that is important in both degradation and 3'-processing of eukaryotic RNAs. We present the crystal structure of the Rrp40 exosome subunit from Saccharomyces cerevisiae at a resolution of 2.2 A. The structure comprises an S1 domain and an unusual KH (K homology) domain. Close packing of the S1 and KH domains is stabilized by a GxNG sequence, which is uniquely conserved in exosome KH domains. Nuclear magnetic resonance data reveal the presence of a manganese-binding site at the interface of the two domains. Isothermal titration calorimetry shows that Rrp40 and archaeal Rrp4 alone have very low intrinsic affinity for RNA. The affinity of an archaeal core exosome for RNA is significantly increased in the presence of the S1-KH subunit Rrp4, indicating that multiple subunits might contribute to cooperative binding of RNA substrates by the exosome.

A dual parameter FRET probe for measuring PKC and PKA activity in living cells.

Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.

Genetically encoded FRET probe for PKC activity based on pleckstrin.

We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.