Requires ATP Hydrolysis Research Articles

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Glucose metabolism in skeletal cells

The mammalian skeleton is integral to whole body physiology with a multitude of functions beyond mechanical support and locomotion, including support of hematopoiesis, mineral homeostasis and potentially other endocrine roles. Formation of the skeleton begins in the embryo and mostly from a cartilage template that is ultimately replaced by bone through endochondrial ossification. Skeletal development and maturation continue after birth in most species and last into the second decade of postnatal life in humans. In the mature skeleton, articular cartilage lining the synovial joint surfaces is vital for bodily movement and damages to the cartilage are a hallmark of osteoarthritis. The mature bone tissue undergoes continuous remodeling initiated with bone resorption by osteoclasts and completed with bone formation from osteoblasts. In a healthy state, the exquisite balance between bone resorption and formation is responsible for maintaining a stable bone mass and structural integrity, while meeting the physiological needs for minerals via controlled release from bone. Disruption of the balance in favor of bone resorption is the root cause for osteoporosis. Whereas osteoclasts pump molar quantities of hydrochloric acid to dissolve the bone minerals in a process requiring ATP hydrolysis, osteoblasts build bone mass by synthesizing and secreting copious amounts of bone matrix proteins. Thus, both osteoclasts and osteoblasts engage in energy-intensive activities to fulfill their physiological functions, but the bioenergetics of those and other skeletal cell types are not well understood. Nonetheless, the past ten years have witnessed a resurgence of interest in studies of skeletal cell metabolism, resulting in an unprecedented understanding of energy substrate utilization and its role in cell fate and activity regulation. The present review attempts to synthesize the current findings of glucose metabolism in chondrocytes, osteoblasts and osteoclasts. Advances with the other relevant cell types including skeletal stem cells and marrow adipocytes will not be discussed here as they have been extensively reviewed recently by others (van Gastel and Carmeliet, 2021). Elucidation of the bioenergetic mechanisms in the skeletal cells is likely to open new avenues for developing additional safe and effective bone therapies.

The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

Structural Characterization of Human Heat Shock Protein 90 N-Terminal Domain and Its Variants K112R and K112A in Complex with a Potent 1,2,3-Triazole-Based Inhibitor.

Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.

Reconstitution of the calcium‐triggered contractile apparatus of ciliated protozoa

Protists, notably ciliated protozoa, exhibit a novel form of contraction that is rapidly triggered by calcium ions but does not require ATP hydrolysis. Ultrafast forms of calcium‐triggered contraction had previously been described for the Vorticella stalk and the Spirostomum cell body. In these ciliates, cell structures (stalk or body) contract within a few milliseconds. Spasmin, an EF‐hand calcium binding contractile protein was identified as the key component of the Vorticella spasmoneme, the contractile organelle present within its stalk. In heterotrich ciliates, such as Stentor and Spirostomum, contraction is mediated by branching, spindle‐shaped structures called myonemes, which contain centrin‐like proteins. Bioinformatics analysis of published and recently obtained data revealed the existence of several distinct groups of EF‐hand calcium‐binding contractile proteins in ciliated protozoa. Synthetic genes encoding these calcium‐binding proteins, as well as their putative scaffold proteins, have been engineered for expression in bacteria. In vitro reconstitutions of these contractile systems were then attempted using purified proteins. Tcb2 protein from Tetrahymena thermophila was identified as a major component of contractile gels formed when calcium ions were added to a low ionic strength alkaline extract of the Tetrahymena membrane‐associated cytoskeleton. Purified Tcb2 protein showed calcium‐dependent reversible assembly in microscopic, spectroscopic, and pelleting assays. Tcb2 has also been shown to interact with and drive the collapse of gels formed with either of two other alpha‐helical coiled‐coil cytoskeletal proteins, Epc1 and Fen1. Through proteomic analysis of Spirostomum cytoskeletal preparations, a family of EF‐hand proteins closely related to the calcium‐binding protein centrin have been identified. In addition, a putative centrin‐binding Sfi1‐like scaffold protein has been identified in Spirostomum. Proteins encoding putative components of the Spirostomum ambiguum myoneme contractile systems have been expressed in bacteria and purified. Solutions of these Spirostomum proteins responded to calcium ions in a similar way to that seen for Tetrahymena contractile proteins. Our immediate goal is to engineer Spirostomum contractile proteins to fully reconstitute in vitro the ultrafast myoneme contraction taking place in living Spirostomumcells. These studies should yield insight into the structure, mechanism and regulation of the calcium‐triggered contractile systems present in protistan cells. As a parallel goal, we are working to engineer optically controlled contractile elements built from these proteins, and introduce them into metazoan animal cells and synthetic cells, so that we can manipulate their behaviors through patterned light activation.

Some assembly required: structural and mechanistic studies of histone chaperones, nucleosome assembly factors and ATP dependent chromatin remodelers

All eukaryotes organize their DNA into nucleosomes, consisting of an octamer of the four core histone proteins H2A, H2B, H3, and H4, around which 147 base pairs of DNA are wrapped in two tight superhelical turns. Nucleosomes pack into higher order structures and require histone chaperones and ATP‐dependent remodelers to make the DNA accessible. I will discuss our recent progress in understanding the higher order structure of chromatin at the centromere, and the role of centromere‐binding proteins in locally folding the chromatin fiber. I will also discuss mechanisms by which nucleosomes are remodeled and assembled in an ATP dependent and ATP‐independent manner. The histone chaperone FACT binds and stabilizes partially disassembled nucleosomes by employing an acidic C‐terminal domain as a DNA mimic to clamp down a histone dimer. The chromatin remodeler SMARCAD1 requires ATP hydrolysis to remove histones from a nucleosome, which it then can deposit onto a new DNA segment in an ATP‐independent mechanism, and I will present biochemical and structural evidence for this mode of action.

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Developing an Intensity-Based Conformational Sensor for Snares

Vesicle trafficking, a process essential to organelle biogenesis and secretion, requires the fusion of membranes. Most cellular membrane fusion occurs via the machinery of soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) proteins, whose zippering catalyze the fusion of two opposing membranes. After the fusion event, the zippered (cis-)SNAREs are then disassembled by NSF in a step requiring ATP hydrolysis. To gain insights into the regulatory mechanism of SNARE assembly and disassembly, we engineered an intensity-based conformational sensor for SNAREs (icsenSNARE) by linking the structural transition of the SNARE complex to a fluorescence signal of cpGFP.

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of ‘poisons’, namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

Real-time investigation of the roles of ATP hydrolysis by UvrA and UvrB during DNA damage recognition in nucleotide excision repair

Nucleotide excision repair (NER) stands out among other DNA repair systems for its ability to process a diverse set of unrelated DNA lesions. In bacteria, NER damage detection is orchestrated by the UvrA and UvrB proteins, which form the UvrA2-UvrB2 (UvrAB) damage sensing complex. The highly versatile damage recognition is accomplished in two ATP-dependent steps. In the first step, the UvrAB complex samples the DNA in search of lesion. Subsequently, the presence of DNA damage is verified within the UvrB-DNA complex after UvrA has dissociated. Although the mechanism of bacterial NER damage detection has been extensively investigated, the role of ATP binding and hydrolysis by UvrA and UvrB during this process remains incompletely understood. Here, we report a pre-steady state kinetics Förster resonance energy transfer (FRET) study of the real-time interaction between UvrA, UvrB, and damaged DNA during lesion detection. By using UvrA and UvrB mutants harboring site-specific mutations in the ATP binding sites, we show for the first time that the dissociation of UvrA from the UvrAB-DNA complex does not require ATP hydrolysis by UvrB. We find that ATP hydrolysis by UvrA is not essential, but somehow facilitates the formation of UvrB-DNA complex, with ATP hydrolysis at the proximal site of UvrA playing a more critical role. Consistent with previous reports, our results indicated that the ATPase activity of UvrB is essential for the formation of UvrB-DNA complex but is not required for the binding of the UvrAB complex to DNA.

Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3.

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are 'clamped' in a sub-compartment created by Scc2's association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation.

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.

Single-molecule approach for studying RNAP II transcription initiation using magnetic tweezers.

The initiation of transcription underlies the ability of cells to modulate genome expression as a function of both internal and external signals and the core process of initiation has features that are shared across all domains of life. Specifically, initiation can be sub-divided into promoter recognition, promoter opening, and promoter escape. However, the molecular players and mechanisms used are significantly different in Eukaryotes and Bacteria. In particular, bacterial initiation requires only the formation of RNA polymerase (RNAP) holoenzyme and proceeds as a series of spontaneous conformational changes while eukaryotic initiation requires the formation of the 31-subunit pre-initiation complex (PIC) and often requires ATP hydrolysis by the Ssl2/XPB subunit of the general transcription factor TFIIH. Our mechanistic view of this process in Eukaryotes has recently been improved through a combination of structural and single-molecule approaches which are providing a detailed picture of the structural dynamics that lead to the production of an elongation competent RNAP II and thus, an RNA transcript. Here we provide the methodological details of our single-molecule magnetic tweezers studies of transcription initiation using purified factors from Saccharomyces cerevisiae.

Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

To repair DNA double-strand breaks by homologous recombination, the 5'-terminated DNA strands must first be resected to produce 3' overhangs. Mre11 from Saccharomyces cerevisiae is a 3' → 5' exonuclease that is responsible for 5' end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5'-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3' end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3' → 5' exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5'-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3' → 5' degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5'-terminated DNA.

Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP.

Mutagenic translesion DNA polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.

Probing the role of Arg97 in Heat shock protein 90 N-terminal domain from the parasite Leishmania braziliensis through site-directed mutagenesis on the human counterpart

In Brazil, the mucocutaneous form of leishmaniasis, caused by the parasite Leishmania braziliensis, is a widespread and very challenging disease responsible for disfiguration and, in the most severe cases, death. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone playing a pivotal role in the folding process of client proteins, and therefore its activity is fundamental for cell survival and proliferation. Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP binding pocket in the protein N-terminal domain (NTD) act as Hsp90 inhibitors. The development of selective molecules targeting the ATPase site of protozoan Hsp90 is tricky for the high homology with the human Hsp90 NTD (hNTD). Notably, only the human Lys112 is replaced by Arg97 in the L. braziliensis enzyme. Recently, this difference has been probed to design selective inhibitors targeting parasite Hsp90s. Here, a reliable protocol for expression and purification of LbHsp90-NTD (LbNTD) was developed but its structural characterization was unsuccessful. The role of Arg97 in LbNTD was hence probed by means of the “leishmanized” K112R variant of hNTDα. To deeply investigate the role of this residue, also the hNTDα K112A variant was generated. Structural studies performed on hNTDα and its variants using various ADP and ATP analogues and cAMP revealed that this residue is not crucial for nucleotide binding. This finding strongly suggests that Arg97 in LbNTD and more generally the conserved arginine residue in parasite Hsp90s are not exploitable for the development of selective inhibitors.

Structural basis for disassembly of katanin heterododecamers

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins.

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical modeling to show that the bacterial chaperones GroEL (Hsp60) and DnaK (Hsp70) both use part of the energy from ATP hydrolysis to restore the native state of their substrates, even under denaturing conditions in which the native state is thermodynamically unstable. Consistently with thermodynamics, upon exhaustion of ATP, the metastable native chaperone products spontaneously revert to their equilibrium non-native states. In the presence of ATPase chaperones, some proteins may thus behave as open ATP-driven, nonequilibrium systems whose fate is only partially determined by equilibrium thermodynamics.

Sec17/Sec18 act twice, enhancing membrane fusion and then disassembling cis-SNARE complexes.

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.

Alpha 2 Na+,K+-ATPase silencing induces loss of inflammatory response and ouabain protection in glial cells

Ouabain (OUA) is a cardiac glycoside that binds to Na+,K+-ATPase (NKA), a conserved membrane protein that controls cell transmembrane ionic concentrations and requires ATP hydrolysis. At nM concentrations, OUA activates signaling pathways that are not related to its typical inhibitory effect on the NKA pump. Activation of these signaling pathways protects against some types of injury of the kidneys and central nervous system. There are 4 isoforms of the alpha subunit of NKA, which are differentially distributed across tissues and may have different physiological roles. Glial cells are important regulators of injury and inflammation in the brain and express the α1 and α2 NKA isoforms. This study investigated the role of α2 NKA in OUA modulation of the neuroinflammatory response induced by lipopolysaccharide (LPS) in mouse primary glial cell cultures. LPS treatment increased lactate dehydrogenase release, while OUA did not decrease cell viability and blocked LPS-induced NF-κB activation. Silencing α2 NKA prevented ERK and NF-κB activation by LPS. α2 NKA also regulates TNF-α and IL-1β levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes.