The AAA+ ATPase p97, a cellular multitool.

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

Forkhead-associated (FHA) domain containing ABC transporter Rv1747 is positively regulated by Ser/Thr phosphorylation in Mycobacterium tuberculosis.

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.

Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients.

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.

C-reactive protein exists in an NaCl concentration-dependent pentamer-decamer equilibrium in physiological buffer.

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca(2+)-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mM Ca(2+) exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s(20,w)(0) of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca(2+), CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R(G) values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K(D) value of 21 microM in solution (140 mM NaCl, 2 mM Ca(2+)) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K(D) value of 23 microM (140 mM NaCl, 2 mM Ca(2+)), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mM Ca(2+) and 140 mM NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.

Novel Selection Approaches to Identify Antibodies Targeting Neoepitopes on the C5b6 Intermediate Complex to Inhibit Membrane Attack Complex Formation.

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein-protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.

Crystal structure of the catalytic D2 domain of the AAA+ ATPase p97 reveals a putative helical split-washer-type mechanism for substrate unfolding.

Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High-resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high-resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by p97. DATABASE: The atomic coordinates and structure factors have been deposited in the PDB database under the accession numbers 6G2V, 6G2W, 6G2X, 6G2Y, 6G2Z and 6G30.

Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa.

Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

An intramolecular switch regulates phosphoindependent FHA domain interactions in Mycobacterium tuberculosis.

Forkhead-associated (FHA) domains have gained considerable prominence as ubiquitous phosphothreonine-dependent binding modules; however, their precise roles in serine and threonine kinase (STK) pathways and mechanisms of regulation remain unclear. From experiments with Rv1827, an FHA domain-containing protein from Mycobacterium tuberculosis, we derived a complete molecular description of an FHA-mediated STK signaling process. First, binding of the FHA domain to each of three metabolic enzyme complexes regulated their catalytic activities but did not require priming phosphorylation. However, phosphorylation of a threonine residue within a conserved amino-terminal motif of Rv1827 triggered its intramolecular association with the FHA domain of Rv1827, thus blocking its interactions with each of the three enzymes. The solution structure of this inactivated form and further mutagenic studies showed how a previously unidentified intramolecular phosphoswitch blocked the access of the target enzymes to a common FHA interaction surface and how this shared surface accommodated three functionally related, but structurally diverse, binding partners. Thus, our data reveal an unsuspected versatility in the FHA domain that allows for the transformation of multiple kinase inputs into various downstream regulatory signals.