Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45^−/− embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45^−/− mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.Ubiquitin-dependent proteolysis is indispensable for various biological processes (3, 40). Protein ubiquitylation is mediated by several enzymes that act in concert, with a ubiquitin ligase (E3) playing a key role in substrate recognition (14). E3 enzymes contain specific structural motifs that mediate recruitment of a ubiquitin-conjugating enzyme (E2), with these motifs including HECT, RING finger, U-box, and PHD finger domains (30). The SCF complex consists of Skp1 (adaptor subunit), Cul1 (scaffold subunit), an F-box protein (substrate recognition subunit), and Rbx1 (also known as Roc1 or Hrt1; RING finger-containing subunit). Whereas Skp1, Cul1, and Rbx1 are common to all SCF complexes, the F-box protein is variable (with ∼70 such proteins having been identified in humans) and confers substrate specificity.Fbxo45 is an F-box protein that was originally isolated as an estrogen-induced protein (47). Human and mouse Fbxo45 genes comprise three exons and possess several consensus binding sequences for the estrogen receptor in the promoter region. Fbxo45 mRNA is rapidly induced on exposure of MCF-7 cells to 17β-estradiol (47). FSN-1, the Caenorhabditis elegans ortholog of Fbxo45, binds to RPM-1 (regulator of presynaptic morphology 1) together with CUL-1 and SKR-1, the C. elegans orthologs of mammalian Cul1 and Skp1, respectively (21, 46). RPM-1 belongs to an evolutionarily conserved family of proteins (the PHR family) that include Highwire (HIW) (Drosophila melanogaster), Esrom (Danio rerio), Phr1 (Mus musculus), and protein associated with Myc (PAM) (Homo sapiens), each of which contains a RING-finger domain that is required for its E3 activity (7, 20, 21, 27, 44). Complete loss of function of fsn-1 in C. elegans results in defects that are characterized by the simultaneous presence of overdeveloped and underdeveloped neuromuscular junctions (NMJs) and which are similar to, but not as pronounced as, those observed in rpm-1^−/− mutants. These genetic findings support the notion that the functions of FSN-1 and RPM-1 are partially overlapping (21).Although PHR family members interact with many potential targets (11, 24, 26, 31), genetic data have shown that one key substrate of RPM-1 and HIW is the mitogen-activated protein kinase kinase kinase known as DLK (dual leucine zipper kinase) in C. elegans and known as Wallenda in D. melanogaster, respectively. The abundance of this kinase is increased in rpm-1 or hiw mutants, and synaptic defects in the mutant worms and flies are suppressed by a loss of DLK or Wallenda. Furthermore, an increase in the level of DLK or Wallenda is sufficient to phenocopy the synaptic defects of the rpm-1 or hiw mutants (5, 27). PAM has also been shown to catalyze the ubiquitylation of tuberin (TSC2) and to regulate signaling by mTOR (mammalian target of rapamycin) in human cells (12).To elucidate the physiological functions of Fbxo45 in mammals, we have now generated mice deficient in this protein. Analysis of the mutant mice revealed that Fbxo45 is required for normal neuromuscular synaptogenesis, axon pathfinding, and neuronal migration. Moreover, we found that Fbxo45 does not form an authentic SCF complex as a result of an amino acid substitution in the F-box domain, and we identified PAM as a binding partner of Fbxo45. The phenotype of Fbxo45^−/− mice was found to be similar to that of Phr1^−/− mice, especially with regard to the defects of neuromuscular synapse formation and of axon navigation. Our results indicate that three fundamental processes of neural development— axonal projection, synapse formation, and neuronal migration—may be linked by a common machinery consisting of the Fbxo45-Phr1 complex.     

HERC1 Ubiquitin Ligase Is Required for Normal Axonal Myelination in the Peripheral Nervous System

A missense mutation in HERC1 provokes loss of cerebellar Purkinje cells, tremor, and unstable gait in tambaleante (tbl) mice. Recently, we have shown that before cerebellar degeneration takes place, the tbl mouse suffers from a reduction in the number of vesicles available for release at the neuromuscular junction (NMJ). The aim of the present work was to study to which extent the alteration in HERC1 may affect other cells in the nervous system and how this may influence the motor dysfunction observed in these mice. The functional analysis showed a consistent delay in the propagation of the action potential in mutant mice in comparison with control littermates. Morphological analyses of glial cells in motor axons revealed signs of compact myelin damage as tomacula and local hypermyelination foci. Moreover, we observed an alteration in non-myelinated terminal Schwann cells at the level of the NMJ. Additionally, we found a significant increment of phosphorylated Akt-2 in the sciatic nerve. Based on these findings, we propose a molecular model that could explain how mutated HERC1 in tbl mice affects the myelination process in the peripheral nervous system. Finally, since the myelin abnormalities found in tbl mice are histological hallmarks of neuropathic periphery diseases, tbl mutant mice could be considered as a new mouse model for this type of diseases.     

Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.     

The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box–type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP-triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs, suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP-induced signaling.     

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.Mind bomb 1 (Mib1)¹, an E3 ubiquitin ligase, is a critical regulator of metazoan development with a large, and ever expanding, number of functions through interactions with a variety of protein partners. Mib1 mutants were first found in zebrafish mutagenesis screens (1), in which the mutants had neurogenic defects, most notably supernumerary primary neurons, and additional deficits in the development of somites (2), ear (3), and vasculature (4). These phenotypes are predominantly the consequences of impaired Notch signaling, as Mib1 is an essential activator of Notch Delta/Serrate/lag-2 (DSL) ligands (1). Mib1 also controls the development of several other organ and tissue systems, including gastrointestinal tract (5), limb bud (6), and the immune system (7). Mib1 is highly conserved across species. For instance, zebrafish Mib1 protein is 68%, 94%, and 94% identical to its fly, mouse, and human orthologs, respectively (8). Moreover, Mib1 has a paralog (Mib2) that shares 38% identical protein sequence with Mib1 in mouse (9). Mib2 is only abundantly expressed in adult tissue, however, and thus does not function in early development. Consistently, Mib1 knockout in mice results in embryonic mortality (10), whereas Mib2 deletion has no obvious effect on mouse development (6).In addition to its role in cell fate determination during early development, Mib1 is also abundantly expressed in the adult brain (11) and plays an important role in neuronal morphogenesis (12). Neurons usually have two basic polarized structures, a single extended axon for sending signals and multiple branched dendrites (or more precisely, the somatodendritic compartment) for receiving signals. Many principle neurons in mammals further grow dendritic spines that are tiny protrusions extended from dendritic branches, creating local postsynaptic compartments for the formation of excitatory synapses. In these synapses, the postsynaptic density (PSD) is an electron-dense membrane thickening aligned with the presynaptic active zone at synaptic junctions. During neuronal morphogenesis, axonal growth and path finding (13), dendrite formation (14), dendritic spine assembly (15), and synaptogenesis (16) are independent but highly related processes controlled by genetic elements and environmental cues. Although dramatic progress has been made in identifying the signaling cascades responsible for these processes, large gaps still remain in the connection of individual signaling components as well as in the coordination of multiple pathways. Our previous proteomics analysis identified that Mib1 is highly enriched in the PSD fraction, and regulates neurite outgrowth in postmitotic neurons (12). Mib1 conditional knockout mouse studies suggest a role in long-term potentiation (LTP) and synaptic plasticity (11), and further intriguing actions of Mib1 continue to be discovered. Mib1 was found to mediate the degradation of survival motor neuron 1 (SMN1), which contributes to spinal muscular atrophy (17). Mib1 was reported to be essential for Wnt3A activation of beta-catenin signaling through the receptor RYK (18), and a recent yeast two-hybrid screen indicated that Mib1 interacts with 81 candidate proteins beyond the canonical Notch pathway (19). The ongoing identification of new Mib1 interaction partners and functions underscores the need to characterize the Mib1 interactome en masse with high confidence.The combination of affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful method for analyzing protein interaction networks. Technological advances in LC-MS/MS have continually increased the sensitivity of protein detection (20, 21), allowing for the analysis of complex samples (22). The primary advantage of this technique, however, has also proven to be its greatest weakness: without stringent washes and data filtering, a vast number of false positives are included in the resulting data sets (23). Methods such as tandem-affinity purification (24) have been developed to remove nonspecific contaminants, but two-step purification requires large quantities of starting materials and reduces sensitivity to loosely bound proteins. Removing contaminants by buffers containing high concentrations of salt and detergents can help limit false positives, but a delicate balance lies between rinsing contaminants and losing weakly bound but true interaction partners, and thus inflating false negative results. In addition, in vivo crosslinking and quantitative analysis are used to enhance the capture of transient interacting proteins (25, 26).To this end, we attempted to characterize the Mib1 interactome by combining glutathione S-transferase (GST) protein affinity purification and advanced quantitative mass spectrometry. In our sequential elution strategy, Mib1 interaction partners were bound to affinity resins coated with GST-Mib1 domains, then eluted in three sequential buffers of increasing stringency. Proteins in these three eluents were identified and quantified by an isobaric labeling Tandem Mass Tag (TMT) method (15). The elution profile of each protein reflected its binding affinity to the GST-Mib1 resins. The strategy not only provides high sensitivity to recover weakly bound partners, but also allows for the affinity-based classification of the interactome and the removal of contaminants. By this approach, we were able to recover 817 putative Mib1 binding partners in adult rat brain and accepted about half of the proteins with high confidence. This study also uncovered that Mib1 interacts with CDKL5, a protein kinase implicated in early infantile epileptic encephalopathy-2 (EIEE2), a severe form of epilepsy and mental retardation in females (28). We then found that Mib1 acts to down-regulate CDKL5 and inhibits its promotion of dendritic spine outgrowth.     

Ubiquitin E3 Ligase A20 is Required in Degradation of Microbial Superantigens in Vascular Endothelial Cells

The endothelial cells and tight junctions or adherens junctions form the endothelial barrier on the inner surface of the blood vessels. How the endothelial barrier degrades the endocytic microbial products, such as Staphylococcal enterotoxin B (SEB), is not fully understood yet. Ubiquitination is involved in protein degradation. This study aims to investigate the role of ubiquitin E3 ligase A20 (A20) in the degradation of endocytic SEB in endothelial cells. The human microvascular endothelial cell line, Hmvec, was cultured to monolayers in the inserts of transwells. SEB was added to the apical chambers to observe the endocytosis and degradation of SEB in Hmvecs. The fusion of endosome/lysosome was observed by immune staining. After exposed to SEB for 30 min, SEB was detected in Hmvecs. SEB could attach to the surface of Hmvecs and endocytosed into the cytoplasm of Hmvecs. The endocytosed SEB was degraded in the Hmvecs, which was transported to the transwell basal chambers in A20-deficient Hmvec monolayers. The SEB-carrying endosomes fused to the lysosomes in Hmvecs; the fusion of endosome/lysosome was disturbed in A20-deficient Hmvecs. In conclusion, A20 plays an important role in the degradation of the endocytic microbial product, SEB, in cardiac endothelial cells.     

Deficiency for the Ubiquitin Ligase UBE3B in a Blepharophimosis-Ptosis-Intellectual-Disability Syndrome

Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.     

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.     

Cytosolic Iron-Sulfur Assembly Is Evolutionarily Tuned by a Cancer-Amplified Ubiquitin Ligase

1. Download high-res image (226KB)
2. Download full-size image

     

Structure of an SspH1-PKN1 Complex Reveals the Basis for Host Substrate Recognition and Mechanism of Activation for a Bacterial E3 Ubiquitin Ligase

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Crystal Structure of the S. solfataricus Archaeal Exosome Reveals Conformational Flexibility in the RNA-Binding Ring

Background

The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/Principal Findings

Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/Significance

This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.     

Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson''s disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin''s enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.     

Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.