Phyllis I. Hanson

AAA+ proteins: have engine, will work.

The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The familys defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.

Calmodulin trapping by calcium-calmodulin-dependent protein kinase

Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.

Membrane budding and scission by the ESCRT machinery: it's all in the neck

The endosomal sorting complexes required for transport (ESCRTs) catalyse one of the most unusual membrane remodelling events in cell biology. ESCRT-I and ESCRT-II direct membrane budding away from the cytosol by stabilizing bud necks without coating the buds and without being consumed in the buds. ESCRT-III cleaves the bud necks from their cytosolic faces. ESCRT-III-mediated membrane neck cleavage is crucial for many processes, including the biogenesis of multivesicular bodies, viral budding, cytokinesis and, probably, autophagy. Recent studies of ultrastructures induced by ESCRT-III overexpression in cells and the in vitro reconstitution of the budding and scission reactions have led to breakthroughs in understanding these remarkable membrane reactions.

The AMPA Receptor GluR2 C Terminus Can Mediate a Reversible, ATP-Dependent Interaction with NSF and α- and β-SNAPs

In this study, we demonstrate specific interaction of the GluR2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide–sensitive fusion protein (NSF) and α- and β-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2–NSF–SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2–NSF–SNAP complex is similar to that of the t-SNARE syntaxin–NSF–SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

The pathophysiological basis of dystonias

Dystonias comprise a group of movement disorders that are characterized by involuntary movements and postures. Insight into the nature of neuronal dysfunction has been provided by the identification of genes responsible for primary dystonias, the characterization of animal models and functional evaluations and in vivo brain imaging of patients with dystonia. The data suggest that alterations in neuronal development and communication within the brain create a susceptible substratum for dystonia. Although there is no overt neurodegeneration in most forms of dystonia, there are functional and microstructural brain alterations. Dystonia offers a window into the mechanisms whereby subtle changes in neuronal function, particularly in sensorimotor circuits that are associated with motor learning and memory, can corrupt normal coordination and lead to a disabling motor disorder.

Plasma membrane deformation by circular arrays of ESCRT-III protein filaments.

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used “deep-etch” electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis–deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.

Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase which primes and/or dissociates SNARE complexes involved in intracellular fusion events. Each NSF protomer contains three domains: an N-terminal domain required for SNARE binding and two ATPase domains, termed D1 and D2, with D2 being required for oligomerization. We have determined the 1.9 Å crystal structure of the D2 domain of NSF complexed with ATP using multi-wavelength anomalous dispersion phasing. D2 consists of a nucleotide binding subdomain with a Rossmann fold and a C-terminal subdomain, which is structurally unique among nucleotide binding proteins. There are interactions between the ATP moiety and both the neighboring D2 protomer and the C-terminal subdomain that may be important for ATP-dependent oligomerization. Of particular importance are three well-ordered and conserved lysine residues that form ionic interactions with the β- and γ-phosphates, one of which likely contributes to the low hydrolytic activity of D2.

Multivesicular body morphogenesis.

Multivesicular bodies (MVBs) are unique organelles in the endocytic pathway that contain vesicles in their lumen. Sorting and incorporation of material into such vesicles is a critical cellular process that has been intensely studied following discovery of the ESCRT (endosomal sorting complex required for transport) machinery just more than a decade ago. In this review, we summarize current understanding of the cellular functions of MVBs and how the ESCRT machinery contributes to MVB morphogenesis. We also highlight the importance of MVBs and ESCRTs in human health. We identify critical areas in which further mechanistic and spatiotemporal studies in living cells will advance this exciting area of research.

TDP-43 accumulation in inclusion body myopathy muscle suggests a common pathogenic mechanism with frontotemporal dementia

TAR DNA binding protein-43 (TDP-43) is found in ubiquitinated inclusions (UBIs) in some frontotemporal dementias (FTD-U). One form of FTD-U, due to mutations in the valosin containing protein (VCP) gene, occurs with an inclusion body myopathy (IBMPFD). Since IBMPFD brain has TDP-43 in UBIs, we looked for TDP-43 inclusions in IBMPFD muscle. In normal muscle, TDP-43 is present in nuclei. In IBMPFD muscle, TDP-43 is additionally present as large inclusions within UBIs in muscle cytoplasm. TDP-43 inclusions were also found in 78% of sporadic inclusion body myositis (sIBM) muscles. In IBMPFD and sIBM muscle, TDP-43 migrated with an additional band on immunoblot similar to that reported in FTD-U brains. This study adds sIBM and hereditary inclusion body myopathies to the growing list of TDP-43 positive inclusion diseases.

NSF ATPase and α-/β-SNAPs Disassemble the AMPA Receptor-PICK1 Complex

Abstract AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and α-/β-SNAPs form a complex in the presence of ATPγS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. α- and β-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity.