Biotinylation, a post-translational modification controlled by the rate of protein-protein association

Biotin protein ligases catalyze specific covalent linkage of the coenzyme biotin to biotin-dependent carboxylases. The reaction proceeds in two steps, including synthesis of an adenylated intermediate followed by biotin transfer to the carboxylase substrate. In this work specificity in the transfer reaction was investigated using single turnover stopped-flow and quench-flow assays. Cognate and noncognate reactions were measured using the enzymes and minimal biotin acceptor substrates from Escherichia coli, Pyrococcus horikoshii, and Homo sapiens. The kinetic analysis demonstrates that for all enzyme-substrate pairs the bimolecular rate of association of enzyme with substrate limits post-translational biotinylation. In addition, in noncognate reactions the three enzymes displayed a range of selectivities. These results highlight the importance of protein-protein binding kinetics for specific biotin addition to carboxylases and provide one mechanism for determining biotin distribution in metabolism.     

SYT-SSX1 (synovial sarcoma translocated) regulates PIASy ligase activity to cause overexpression of NCOA3 protein

Chromosomal translocations are a major source of genetic abnormalities causally linked to certain malignancies. Synovial sarcoma is an aggressive soft tissue tumor characterized by a chromosomal translocation between chromosome 18 and X, generating oncoproteins such as SYT-SSX1 and SYT-SSX2. The molecular mechanism underlying the oncogenic potential of SYT-SSX1/2 is not clear. Here we show that SYT-SSX1 leads to up-regulation of NCOA3, a protein critical for the formation of various cancers. The increase of NCOA3 is essential for SYT-SSX1-mediated synovial sarcoma formation. SYT-SSX1 does so by increasing the sumoylation of NCOA3 through interaction with a SUMO E3 ligase, PIASy, as well as the sumoylation of NEMO. NEMO has also been shown to physically interact with NCOA3. Increased sumoylation of NCOA3 leads to its increased steady state level and nuclear localization. Our findings represent the first example that an oncoprotein directly regulates substrate modification by a SUMO E3 ligase, and leads to overexpression of a protein essential for tumor formation. Such a mechanistic finding provides an opportunity to design specific therapeutic interventions to treat synovial sarcoma.     

N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.     

Comparative analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other fic domain-containing enzymes reveals differences in substrate and nucleotide specificities

A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.     

Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.     

Ubiquitin- and MDM2 E3 ligase-independent proteasomal turnover of nucleostemin in response to GTP depletion

Nucleostemin (NS) is a nucleolar GTP-binding protein essential for ribosomal biogenesis, proliferation, and animal embryogenesis. It remains largely unclear how this protein is regulated. While working on its role in suppression of MDM2 and activation of p53, we observed that NS protein (but not mRNA) levels decreased drastically in response to GTP depletion. When trying to further elucidate the molecular mechanism(s) underlying this unusual phenomenon, we found that NS was degraded independently of ubiquitin and MDM2 upon GTP depletion. First, depletion of GTP by treating cells with mycophenolic acid decreased the level of NS without apparently affecting the levels of other nucleolar proteins. Second, mutant NS defective in GTP binding and exported to the nucleoplasm was much less stable than wild-type NS. Although NS was ubiquitinated in cells, its polyubiquitination was independent of Lys-48 or Lys-63 in the ubiquitin molecule. Inactivation of E1 in E1 temperature-sensitive mouse embryonic fibroblast (MEF) cells failed to prevent the proteasomal degradation of NS. The proteasomal turnover of NS was also MDM2-independent, as its half-life in p53/MDM2 double knock-out MEF cells was the same as that in wild-type MEF cells. Moreover, NS ubiquitination was MDM2-independent. Mycophenolic acid or doxorubicin induced NS degradation in various human cancerous cells regardless of the status of MDM2. Hence, these results indicate that NS undergoes a ubiquitin- and MDM2-independent proteasomal degradation when intracellular GTP levels are markedly reduced and also suggest that ubiquitination of NS may be involved in regulation of its function rather than stability.     

Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K_D ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.     

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol⁻¹) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K_D = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K_D) and the regulation of cysteine sensitivity of SAT within the CSC.     

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.     

Alpha-actinin-4 and CLP36 protein deficiencies contribute to podocyte defects in multiple human glomerulopathies

Genetic alterations of α-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-α-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-α-actinin-4 function in human glomerular diseases remains elusive. Here we show that α-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of α-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of α-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated α-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between α-actinin-4 and CLP36. Inhibition of the α-actinin-4-CLP36 complex, like loss of α-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the α-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the α-actinin-4-CLP36 complex in podocytes and provide an explanation as to how α-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a loss-of-function mechanism.     

The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells

Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways.     

Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors

Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.     

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.