Proteomics in the study of hippocampal plasticity

Synaptic plasticity is the dynamic regulation of the strength of synaptic communication between nerve cells. It is central to neuronal development as well as experience-dependent remodeling of the adult nervous system as occurs during memory formation. Aberrant forms of synaptic plasticity also accompany a variety of neurological and psychiatric diseases, and unraveling the biological basis of synaptic plasticity has been a major goal in neurobiology research. The biochemical and structural mechanisms underlying different forms of synaptic plasticity are complex, involving multiple signaling cascades, reconfigurations of structural proteins and the trafficking of synaptic proteins. As such, proteomics should be a valuable tool in dissecting the molecular events underlying normal and disease-related forms of plasticity. In fact, progress in this area has been disappointingly slow. We discuss the particular challenges associated with proteomic interrogation of synaptic plasticity processes and outline ways in which we believe proteomics may advance the field over the next few years. We pay particular attention to technical advances being made in small sample proteomics and the advent of proteomic imaging in studying brain plasticity.     

MEKK4 sequesters RIP2 to dictate NOD2 signal specificity

The Crohn's-disease-susceptibility protein, NOD2, coordinates signaling responses upon intracellular exposure to bacteria. Although NOD2 is known to activate NFkappaB, little is known about the molecular mechanisms by which NOD2 coordinates functionally separate signaling pathways such as NFkappaB, JNK, and p38 to regulate cytokine responses. Given that one of the characteristics of Crohn's disease is an altered cytokine response to normal bacterial flora, the coupling of signaling pathways could be important for Crohn's-disease pathophysiology. We find that a MAP3K, MEKK4, binds to RIP2 to sequester RIP2 from the NOD2 signaling pathway. This MEKK4:RIP2 complex dissociates upon exposure to the NOD2 agonist, MDP, allowing NOD2 to bind to RIP2 and activate NFkappaB. MEKK4 thus sequesters RIP2 to inhibit the NOD2:RIP2 complex from activating NFkappaB signaling pathways, and Crohn's-disease-associated NOD2 polymorphisms cannot compete with MEKK4 for RIP2 binding. Lastly, we find that MEKK4 helps dictate signal specificity downstream of NOD2 activation as knockdown of MEKK4 in macrophages exposed to MDP causes increased NFkappaB activity, absent p38 activity, and hyporesponsiveness to TLR2 and TLR4 agonists. These biochemical findings suggest that basal inhibition of the NOD2-driven NFkappaB pathway by MEKK4 could be important in the pathogenesis of Crohn's disease.     

The yeast Shu complex couples error-free post-replication repair to homologous recombination

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis has been well characterized, little is known about the molecular events involved in error-free bypass, although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide synthetic genetic array screen for novel genes involved in error-free PRR and observed evidence of genetic interactions between error-free PRR and HR. Furthermore, this screen identified and assigned four genes, CSM2 , PSY3 , SHU1 and SHU2 , whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any of these genes is able to suppress the severe phenotypes of top3 and sgs1 . We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1 . Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. This mechanism appears to be conserved throughout eukaryotes.     

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V₁ and membrane V₀ sectors inactivates MgATPase activity of the V₁-ATPase. This inactivation requires the V₁ H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761–21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V₁ and V₀ subunits were identified by two-hybrid assay. The B subunit of the V₁ catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V₁ subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V₀ subunit Vph1p. V₁-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V₁ when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V₁ lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V₁ was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V₁ complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V₁-ATPase activity and precludes V₀ interactions.V-ATPases are ubiquitous proton pumps responsible for compartment acidification in all eukaryotic cells (1, 2). These pumps couple hydrolysis of cytosolic ATP to proton transport into the lysosome/vacuole, endosomes, Golgi apparatus, clathrin-coated vesicles, and synaptic vesicles. Through their role in organelle acidification, V-ATPases are linked to cellular functions as diverse as protein sorting and targeting, zymogen activation, cytosolic pH homeostasis, and resistance to multiple types of stress (3). They are also recruited to the plasma membrane of certain cells, where they catalyze proton export (4, 5).V-ATPases are evolutionarily related to ATP synthases of bacteria and mitochondria and consist of two multisubunit complexes, V₁ and V₀, which contain the sites for ATP hydrolysis and proton transport, respectively. Like the ATP synthase (F-ATPase), V-ATPases utilize a rotational catalytic mechanism. ATP binding and hydrolysis in the three catalytic subunits of the V₁ sector generate sequential conformational changes that drive rotation of a central stalk (6–8). The central stalk subunits are connected to a ring of proteolipid subunits in the V₀ sector that bind protons to be transported. The actual transport is believed to occur at the interface of the proteolipids and V₀ subunit a. Rotational catalysis will be productive in proton transport only if V₀ subunit a is held stationary, whereas the proteolipid ring rotates (8). This “stator function” resides in a single peripheral stalk in F-ATPases (9, 10), but is distributed among up to three peripheral stalks in V-ATPases (11–13). The peripheral stator stalks link V₀ subunit a to the catalytic headgroup and ensures that there is rotation of the central stalk complex relative to the V₀ a subunit and catalytic headgroup.Eukaryotic V-ATPases are highly conserved in both their overall structure and the sequences of individual subunits. Although homologs of most subunits of eukaryotic V-ATPases are present in archaebacterial V-ATPases (also known as A-ATPases), the C and H subunits are unique to eukaryotes. Both subunits have been localized at the interface of the V₁ and V₀ sectors, suggesting that they are positioned to play a critical role in structural and functional interaction between the two sectors (14–16). The yeast C and H subunits are the only eukaryotic V-ATPase subunits for which x-ray crystal structures are available (17, 18). The structure of the C subunit revealed an elongated “dumbbell-shaped” molecule, with foot, head, and neck domains (18). The structure of the H subunit indicated two domains. The N-terminal 348 amino acids fold into a series of HEAT repeats and are connected by a 4-amino acid linker to a C-terminal domain containing amino acids 352–478 (17). These two domains have partially separable functions in the context of the assembled V-ATPase (19). Complexes containing only the N-terminal domain of the H subunit (H-NT)² supported some ATP hydrolysis but little or no proton pumping in isolated vacuolar vesicles (19, 20). The C-terminal domain (H-CT) assembled with the rest of the V-ATPase in the absence of intact subunit H, but supported neither ATPase nor proton pumping activity (19). However, co-expression of the H-NT and H-CT domains results in assembly of both sectors with the V-ATPase and allows increased ATP-driven proton pumping in isolated vacuolar vesicles. These results suggest that the H-NT and H-CT domains play distinct and complementary roles even when the two domains are not covalently attached.In addition to their role as dedicated proton pumps, eukaryotic V-ATPases are also distinguished from F-ATPases and archaeal V-ATPases in their regulation. Eukaryotic V-ATPases are regulated in part by reversible disassembly of the V₁ complex from the V₀ complex (1, 21, 22). In yeast, disassembly of previously assembled complexes occurs in response to glucose deprivation, and reassembly is rapidly induced by glucose readdition to glucose-deprived cells. Disassembly down-regulates pump activity, and both the disassembled sectors are inactivated. Inhibition of ATP hydrolysis in free V₁ sectors is particularly critical, because release of an active ATPase into the cytosol could deplete cytosolic ATP stores. This inhibition is dependent in part on the H subunit. V₁ complexes isolated from vma13Δ mutants, which lack the H subunit gene (V₁(-H) complexes) have MgATPase activity. Consistent with a physiological role for H subunit inhibition of V₁, heterozygous diploids containing elevated levels of free V₁ complexes without subunit H have severe growth defects (23). V₁ complexes containing subunit H have no MgATPase activity, but retain some CaATPase activity, suggesting a role for nucleotides in inhibition (24, 25). Consistent with such a role, both the CaATPase activity of native V₁ and the MgATPase activity of V₁(-H) complexes are lost within a few minutes of nucleotide addition (24).A number of points of interaction between the H subunit and the V₁ and V₀ complexes have been identified through two-hybrid assays, binding of expressed proteins, and cross-linking experiments. These experiments have indicated that the H subunit binds to V₁ subunits E and G of the V-ATPase peripheral stalks (26, 27), the catalytic subunit (V₁ subunit A) (28), regulatory V₁ subunit B (15), and the N-terminal domain of subunit a (28). Recently, Jeffries and Forgac (29) have found that cysteines introduced into the C-terminal domain of subunit H can be cross-linked to subunit F in isolated V₁ sectors via a 10-Å cross-linking reagent.In this work, we examine both the subunit-subunit interactions and functional roles of the H-NT and H-CT domains in inhibition of V₁-ATPase activity. When expressed in yeast cells lacking subunit H, H-NT can be isolated with cytosolic V₁ complexes, but H-CT cannot. We find that both of these domains contribute to inhibition of ATPase activity, but that stable binding to V₁ and full inhibition of activity requires both domains. We also find that the H-CT can bind to the cytosolic N-terminal domain of V₀ subunit Vph1p (Vph1-NT) in isolation, but does not support tight binding of Vph1-NT to isolated V₁ complexes.