Abstract The P2X receptor is an ATP-gated cation channel expressed on the plasma membrane. The head domain (Gln111–Val167 in the rat P2X4 receptor) regulates ATP-induced cation influx. In this study, we prepared a head domain with three disulfide bonds, such as the intact rat P2X4 receptor contains. NMR analysis showed that the head domain possessed the same fold as in the zebrafish P2X4 receptor previously determined by crystallography. Furthermore, the inhibitory, divalent, metal ion binding sites were determined by NMR techniques. These findings will be useful for the design of specific inhibitors for the P2X receptor family.

Abstract The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.

Abstract Reptin and Pontin belong to the AAA+ ATPase family of DNA helicases. Both proteins are present in several chromatin-remodeling machineries and are involved in transcriptional regulation, DNA repair, and telomerase activity, but they also function independently from each other. Here we report the identification of p65 as an interacting partner of Reptin. Using reporter gene assays, we show Reptin inhibits NF-κB transactivation after TNFα stimulation. Reptin is mainly localized in the cytoplasm and impedes NF-κB activation by inhibiting IκB-α degradation and restraining p65 nuclear translocation. These results reveal a novel mechanism for the control of NF-κB pathway by cytoplasmic Reptin.

Abstract The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure–function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

Abstract UNC-51 like kinase (ULK1) translocates to dysfunctional mitochondria and is involved in mitophagy, but the mechanisms responsible for ULK1 activation and translocation remain unclear. Here, we found that hypoxia induces phosphorylation of ULK1 at Serine-555 by Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK). Unlike wild-type ULK1, an ULK1 (S555A) mutant cannot translocate to mitochondria in response to hypoxia. Inhibition or knockdown of AMPK prevents ULK1 translocation and inhibits mitophagy. Finally, the phospho-mimic ULK1 (S555D) mutant, but not ULK1 (S555A), rescues mitophagy in AMPK-knockdown cells. Thus, we conclude that AMPK-dependent phosphorylation of ULK1 is critical for translocation of ULK1 to mitochondria and for mitophagy in response to hypoxic stress.

Abstract Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface.

Abstract Mpn1 is an exoribonuclease that modifies the spliceosomal small nuclear RNA (snRNA) U6 by trimming its oligouridine tail and introducing a cyclic phosphate group (>p). Mpn1 deficiency induces U6 3′ end misprocessing, accelerated U6 decay and pre-mRNA splicing defects. Mutations in the human MPN1 gene are associated with the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Here we present the deep sequencing of the >p-containing transcriptomes of mpn1Δ fission yeast and PN cells. While in yeast U6 seems to be the only substrate of Mpn1, human Mpn1 also processes U6atac snRNA. PN cells bear unstable U6atac species with aberrantly long and oligoadenylated 3′ ends. Our data corroborate the link between Mpn1 and snRNA stability suggesting that PN could derive from pre-mRNA splicing aberrations.

Abstract Glycoside hydrolase family 13 contains exo-glucosidases specific for α-(1 → 4)- and α-(1 → 6)-linkages including α-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The α-(1 → 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to α-(1 → 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher kcat/Km for maltose than wild-type, but its kcat/Km for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher kcat/Km for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the α-(1 → 6)-glucosidic linkage.

Abstract Dexras1 is a small GTPase and plays a central role in neuronal iron trafficking. We have shown that stimulation of glutamate receptors activates neuronal nitric oxide synthase, leading to S-nitrosylation of Dexras1 and a physiological increase in iron uptake. Here we report that Dexras1 is phosphorylated by protein kinase A (PKA) on serine 253, leading to a suppression of iron influx. These effects were directly associated with the levels of S-nitrosylated Dexras1, whereby PKA activation reduced Dexras1 S-nitrosylation in a dose dependent manner. Moreover, we found that adiponectin modulates Dexras1 via PKA. Hence these findings suggest the involvement of the PKA pathway in modulating glutamate-mediated ROS in neurons, and hint to a functional crosstalk between S-nitrosylation and phosphorylation.

Abstract Protein tyrosine phosphatase A (PtpA) has been shown to play a key role in human macrophage infection by Mycobacterium tuberculosis (Mtb). Protein tyrosine kinase A (PtkA) was the first protein tyrosine kinase shown to phosphorylate PtpA. Here, we found that PtkA-mediated phosphorylation of PtPA on Tyr-128 and Tyr-129 enhances the PtPA phosphatase activity. Moreover, ex-vivo protein–protein interaction assays showed that PtpA can be phosphorylated by several eukaryotic-like Ser/Thr protein kinases, such as protein kinase A (PknA). PknA was found to regulate PtpA phosphatase activity through Thr-45 phosphorylation. These results indicate that members of two independent families of protein kinases tune PtpA activity in Mtb.

Abstract The P2X receptor is an ATP-gated cation channel expressed on the plasma membrane. The head domain (Gln111–Val167 in the rat P2X4 receptor) regulates ATP-induced cation influx. In this study, we prepared a head domain with three disulfide bonds, such as the intact rat P2X4 receptor contains. NMR analysis showed that the head domain possessed the same fold as in the zebrafish P2X4 receptor previously determined by crystallography. Furthermore, the inhibitory, divalent, metal ion binding sites were determined by NMR techniques. These findings will be useful for the design of specific inhibitors for the P2X receptor family.

Abstract The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.

Abstract Reptin and Pontin belong to the AAA+ ATPase family of DNA helicases. Both proteins are present in several chromatin-remodeling machineries and are involved in transcriptional regulation, DNA repair, and telomerase activity, but they also function independently from each other. Here we report the identification of p65 as an interacting partner of Reptin. Using reporter gene assays, we show Reptin inhibits NF-κB transactivation after TNFα stimulation. Reptin is mainly localized in the cytoplasm and impedes NF-κB activation by inhibiting IκB-α degradation and restraining p65 nuclear translocation. These results reveal a novel mechanism for the control of NF-κB pathway by cytoplasmic Reptin.

Abstract The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure–function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

Abstract UNC-51 like kinase (ULK1) translocates to dysfunctional mitochondria and is involved in mitophagy, but the mechanisms responsible for ULK1 activation and translocation remain unclear. Here, we found that hypoxia induces phosphorylation of ULK1 at Serine-555 by Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK). Unlike wild-type ULK1, an ULK1 (S555A) mutant cannot translocate to mitochondria in response to hypoxia. Inhibition or knockdown of AMPK prevents ULK1 translocation and inhibits mitophagy. Finally, the phospho-mimic ULK1 (S555D) mutant, but not ULK1 (S555A), rescues mitophagy in AMPK-knockdown cells. Thus, we conclude that AMPK-dependent phosphorylation of ULK1 is critical for translocation of ULK1 to mitochondria and for mitophagy in response to hypoxic stress.

Abstract Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface.

Abstract Mpn1 is an exoribonuclease that modifies the spliceosomal small nuclear RNA (snRNA) U6 by trimming its oligouridine tail and introducing a cyclic phosphate group (>p). Mpn1 deficiency induces U6 3′ end misprocessing, accelerated U6 decay and pre-mRNA splicing defects. Mutations in the human MPN1 gene are associated with the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Here we present the deep sequencing of the >p-containing transcriptomes of mpn1Δ fission yeast and PN cells. While in yeast U6 seems to be the only substrate of Mpn1, human Mpn1 also processes U6atac snRNA. PN cells bear unstable U6atac species with aberrantly long and oligoadenylated 3′ ends. Our data corroborate the link between Mpn1 and snRNA stability suggesting that PN could derive from pre-mRNA splicing aberrations.

Abstract Glycoside hydrolase family 13 contains exo-glucosidases specific for α-(1 → 4)- and α-(1 → 6)-linkages including α-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The α-(1 → 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to α-(1 → 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher kcat/Km for maltose than wild-type, but its kcat/Km for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher kcat/Km for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the α-(1 → 6)-glucosidic linkage.

Abstract Dexras1 is a small GTPase and plays a central role in neuronal iron trafficking. We have shown that stimulation of glutamate receptors activates neuronal nitric oxide synthase, leading to S-nitrosylation of Dexras1 and a physiological increase in iron uptake. Here we report that Dexras1 is phosphorylated by protein kinase A (PKA) on serine 253, leading to a suppression of iron influx. These effects were directly associated with the levels of S-nitrosylated Dexras1, whereby PKA activation reduced Dexras1 S-nitrosylation in a dose dependent manner. Moreover, we found that adiponectin modulates Dexras1 via PKA. Hence these findings suggest the involvement of the PKA pathway in modulating glutamate-mediated ROS in neurons, and hint to a functional crosstalk between S-nitrosylation and phosphorylation.

Abstract Protein tyrosine phosphatase A (PtpA) has been shown to play a key role in human macrophage infection by Mycobacterium tuberculosis (Mtb). Protein tyrosine kinase A (PtkA) was the first protein tyrosine kinase shown to phosphorylate PtpA. Here, we found that PtkA-mediated phosphorylation of PtPA on Tyr-128 and Tyr-129 enhances the PtPA phosphatase activity. Moreover, ex-vivo protein–protein interaction assays showed that PtpA can be phosphorylated by several eukaryotic-like Ser/Thr protein kinases, such as protein kinase A (PknA). PknA was found to regulate PtpA phosphatase activity through Thr-45 phosphorylation. These results indicate that members of two independent families of protein kinases tune PtpA activity in Mtb.