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Aquaporins are a family of ubiquitously expressed transmembrane water channels implicated in a broad range of physiological functions. We have previously reported that aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. Using either genetic deletion or small molecule inhibitor, we show that AQP4 supports T cell receptor mediated activation of both mouse and human T cells. Intact AQP4 is required for optimal T cell receptor (TCR)-related signaling events, including nuclear translocation of transcription factors and phosphorylation of proximal TCR signaling molecules. AQP4 deficiency or inhibition impairs actin cytoskeleton rearrangements following TCR crosslinking, causing inferior TCR polarization and a loss of TCR signaling. Our findings reveal a novel function of AQP4 in T lymphocytes and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation.

Aquaporins (AQPs) are water channels that mediate a variety of biological processes. However, their role in the immune system is poorly understood. We recently reported that AQP4 is expressed by naïve and memory T cells and that AQP4 blockade with a small molecule inhibitor prolongs murine heart allograft survival at least partially through diminishing T cell activation, proliferation and trafficking. The goal of this study was to determine how AQP4 function impacts T cells in the absence of antigen stimulation. AQP4 inhibition transiently reduced the number of circulating CD4+ and CD8+ T cells in naïve non-transplanted mice in the absence of systemic T cell depletion. Adoptive transfer studies demonstrated T cell intrinsic effect of AQP4 inhibition. AQP4 blockade altered T cell gene and protein expression of chemokine receptors S1PR1 and CCR7, and their master regulator KLF-2, and reduced chemotaxis toward S1P and CCL21. Consistent with the in vitro data, in vivo AQP4 inhibition reduced T lymphocyte numbers in the lymph nodes with simultaneous accumulation in the liver. Our findings indicate that blocking AQP4 reversibly alters T lymphocyte trafficking pattern. This information can be explored for the treatment of undesirable immune responses in transplant recipients or in patients with autoimmune diseases.

Background: Cerebral edema after cardiac arrest (CA) is associated with increased mortality and unfavorable outcome in children and adults. Aquaporin-4 mediates cerebral water movement and its absence in models of ischemia improves outcome. We investigated early and selective pharmacologic inhibition of aquaporin-4 in a clinically relevant asphyxial CA model in immature rats in a threshold CA insult that produces primarily cytotoxic edema in the absence of blood brain barrier permeability. Methods: Postnatal day 16–18 Sprague-Dawley rats were studied in our established 9-min asphyxial CA model. Rats were randomized to aquaporin-4 inhibitor (AER-271) vs vehicle treatment, initiated at return of spontaneous circulation. Cerebral edema (% brain water) was the primary outcome with secondary assessments of the neurologic deficit score (NDS), hippocampal neuronal death, and neuroinflammation. Results: Treatment with AER-271 ameliorated early cerebral edema measured at 3 h after CA vs. vehicle treated rats. This treatment also attenuated early NDS. In contrast to rats treated with vehicle after CA, rats treated with AER-271 did not develop significant neuronal death or neuroinflammation as compared to sham. Conclusion: Early post-resuscitation aquaporin-4 inhibition blocks the development of early cerebral edema, reduces early neurologic deficit, and blunts neuronal death and neuroinflammation post-CA.

The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies., Highlights • Cryo EM 3D snapshots of non native Rhodospirillum rubrum RuBisCO bound to E. coli GroEL • Two-lobed substrate density in the binary complex may represent a captured form of an early folding intermediate • The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding. • The RuBisCO density is seen in contact with 3 consecutive GroEL apical domains and one remote apical domain.

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia-reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC-mismatched murine cardiac allografts subjected to 8 hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short-time posttransplantation improves the viability of donor graft cells, diminishes donor-reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte-associated antigen 4-Ig in prolonging survival of 8-hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor-reactive T cells. These results identify AQPs as a promising target for diminishing donor-specific alloreactivity and improving the survival of high-risk organ transplants.

Aquaporin-4 (AQP4) plays an important role in regulating water exchange across the blood-brain barrier (BBB) and brain-cerebrospinal fluid interface. Studies on AQP-4 knockout mice (AQP4-KO) have reported considerable protection from brain edema induced by acute water intoxication and ischemic stroke, identifying AQP4 as a potential target for therapeutic interventions. However, the long-term effects of chronic AQP4 suppression are yet to be elucidated. In the current study, we evaluated the physiological and structural changes in adult AQP4-KO mice using magnetic resonance imaging (MRI) and immunohistochemical analysis. Water exchange across BBB was assessed by tracking an intravenous bolus injection of oxygen-17 (17O) water (H217O) using 17O-MRI. Cerebral blood flow (CBF) was quantified using arterial spin-labeling (ASL) MRI. Capillary density was determined by immunohistochemical staining for glucose transporter-1 (GLUT1). Compared to wildtype control mice, AQP4-KO mice showed a significant reduction in peak and steady-state H217O uptake despite unaltered CBF. Interestingly, a 22% increase in cortical capillary density was observed in AQP4-KO mice. These results suggest that increased cerebral vascularization may be an adaptive response to chronic reduction in water exchange across BBB in AQP4-KO mice.

Cerebral edema in ischemic stroke can lead to increased intracranial pressure, reduced cerebral blood flow and neuronal death. Unfortunately, current therapies for cerebral edema are either ineffective or highly invasive. During the development of cytotoxic and subsequent ionic cerebral edema water enters the brain by moving across an intact blood brain barrier and through aquaporin-4 (AQP4) at astrocyte endfeet. Using AQP4-expressing cells, we screened small molecule libraries for inhibitors that reduce AQP4-mediated water permeability. Additional functional assays were used to validate AQP4 inhibition and identified a promising structural series for medicinal chemistry. These efforts improved potency and revealed a compound we designated AER-270, N-[3,5-bis (trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide. AER-270 and a prodrug with enhanced solubility, AER-271 2-{[3,5-Bis(trifluoromethyl) phenyl]carbamoyl}-4-chlorophenyl dihydrogen phosphate, improved neurological outcome and reduced swelling in two models of CNS injury complicated by cerebral edema: water intoxication and ischemic stroke modeled by middle cerebral artery occlusion.

Arterial spin labeling (ASL) is a valuable non-contrast perfusion MRI technique with numerous clinical applications. Many previous ASL MRI studies have utilized either echo-planar imaging (EPI) or true fast imaging with steady-state free precession (true FISP) readouts, which are prone to off-resonance artifacts on high-field MRI scanners. We have developed a rapid ASL-FISP MRI acquisition for high-field preclinical MRI scanners providing perfusion-weighted images with little or no artifacts in less than 2 s. In this initial implementation, a flow-sensitive alternating inversion recovery (FAIR) ASL preparation was combined with a rapid, centrically encoded FISP readout. Validation studies on healthy C57/BL6 mice provided consistent estimation of in vivo mouse brain perfusion at 7 and 9.4 T (249 ± 38 and 241 ± 17 mL/min/100 g, respectively). The utility of this method was further demonstrated in the detection of significant perfusion deficits in a C57/BL6 mouse model of ischemic stroke. Reasonable kidney perfusion estimates were also obtained for a healthy C57/BL6 mouse exhibiting differential perfusion in the renal cortex and medulla. Overall, the ASL-FISP technique provides a rapid and quantitative in vivo assessment of tissue perfusion for high-field MRI scanners with minimal image artifacts. Copyright © 2014 John Wiley & Sons, Ltd.

Aquaporins (AQPs) are membrane channels that mediate a variety of biological processes via water and other small molecule movement. Several aquaglyceroporins have been shown to regulate key immune functions. However, the role of AQP4 in immune cells is poorly characterized. We recently reported that AQP4 is expressed by both naïve and memory CD4 and CD8 T cells. Furthermore, AQP4 blockade with a small molecule inhibitor, AER-270, reduced T cell activation and proliferation leading to a significant prolongation of fully MHC-mismatched murine heart allograft survival. The goal of this study was to determine the mechanisms by which AQP4 facilitates T cell activation and TCR signaling. Wild-type (WT) but not AQP4−/− T cells treated with AER-270 were protected from lysis in a hypoosmotic shock assay demonstrating AQP4 requirement for water transport in T cells. Following αCD3/αCD28 stimulation, AQP4−/− T cells demonstrated inferior activation compared to WT T cells. Similar decreased activation was observed after treatment of WT T cells with AER-270. Consistent with reduced T cell activation, AQP4 inhibition diminished Ca2+flux in WT but not AQP4−/− T cells in response to TCR crosslinking. AQP4 inhibition also resulted in altered pattern of protein tyrosine phosphorylation at 2 min following TCR crosslinking. These results indicate that despite T cell expression of several water channels, AQP4 function is critical for optimal T cell activation and functions. Our study identifies a novel role of water channels in T cell biology and suggests AQP4 inhibition as a promising strategy to modulate T cell alloresponses following transplantation.

Mutations of cytosolic Cu/Zn superoxide dismutase 1 (SOD1) in humans and overexpression of mutant human SOD1 genes in transgenic mice are associated with the motor neuron degenerative condition known as amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease). Gain-of-function toxicity from the mutant protein expressed in motor neurons, associated with its misfolding and aggregation, leads to dysfunction and cell death, associated with paralyzing disease. Here, using hydrogen–deuterium exchange in intact mice in vivo, we have addressed whether an ALS-associated mutant protein, G85R SOD1–YFP, is subject to the same rate of turnover in spinal cord both early in the course of the disease and later. We find that the mutant protein turns over about 10-fold faster than a similarly expressed wild-type fusion and that there is no significant change in the rate of turnover as animals age and disease progresses.

The water channels aquaporins mediate a variety of immune functions. We previously demonstrated that aquaporin 4 (AQP4) is expressed by CD4 and CD8 T cells and blockade of AQP4 with small molecule inhibitor AER-270/271 significantly prolongs survival of heart allografts in two robust models of rejection. The goal of this study was to determine the effects of AQP4 blockade on T cells. Administration of AER-271 into naïve non-transplanted mice (250 μg i.p. every 6 h on d. 0–4) decreased numbers of circulating CD4 and CD8 T cells, but not B cells, by >90%, compared to untreated controls. The T cell frequencies and numbers in the secondary lymphoid organs (SLOs) were not significantly affected by AER-271, suggesting the lack of circulating T cells is not due to systemic depletion but rather to altered T cell trafficking. The effect of AER-271 treatment was transient, as circulating T cell numbers returned to pretreatment levels by d 21. AER-271 treated animals promptly rejected heart allografts transplanted 24 d after treatment cessation (MST 6 d, n=4). We next tested the effects of AQP4 blockade on Sphingosine-1 Phosphate Receptor 1 (S1PR1), a key mediator of T cell trafficking. S1PR1 mRNA expression was reduced in isolated mouse spleen T cells within 3 h of in vitro incubation with AER-270. Decreased S1PR1 mRNA expression translated into altered chemotaxis, as AER-270 reduced T cell migration toward S1PR1 ligand, Sphingosine-1 Phosphate (S1P), in a transwell system. Our data suggest that AER-270/271 treatment results in altered S1PR1 transcription thus changing T cell trafficking and prolonging allograft survival. Therefore, AQP4 blockade may be an attractive therapeutic strategy in transplantation and other immune-mediated diseases.

Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a “power stroke” of forceful apical movement to eject polypeptide into the chamber.

Summary The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of “dwell time” for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the ≈10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of “close confinement” on folding in the cis cavity.

The double ring chaperonin GroEL and its lid-like co-chaperonin GroES form asymmetric complexes that in the ATP-bound state mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptide and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by electron cryo-microscopy and single particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and of the substrate-binding domains, admitting ATP and new substrate protein.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe 4 S 4 cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe 4 S 4 cofactor formation, independent of ATP and GroES.

The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines.

Cyclin E, a partner of the cyclin-dependent kinase Cdk2, has been implicated in positive control of the G1/S phase transition. Whereas degradation of cyclin E has been shown to be exquisitely regulated by ubiquitination and proteasomal action, little is known about posttranscriptional aspects of its biogenesis. In a yeast-based screen designed to identify human proteins that interact with human cyclin E, we identified components of the eukaryotic cytosolic chaperonin CCT. We found that the endogenous CCT complex in yeast was essential for the maturation of cyclin E in vivo. Under conditions of impaired CCT function, cyclin E failed to accumulate. Furthermore, newly translated cyclin E, both in vitro in reticulocyte lysate and in vivo in human cells in culture, is efficiently bound and processed by the CCT. In vitro, in the presence of ATP, the bound protein is folded and released in order to become associated with Cdk2. Thus, both the acquisition of the native state and turnover of cyclin E involve ATP-dependent processes mediated by large oligomeric assemblies.

Protein folding by the double-ring chaperonin GroEL is initiated in cis ternary complexes, in which polypeptide is sequestered in the central channel of a GroEL ring, capped by the co-chaperonin GroES. The cis ternary complex is dissociated (half-life of approximately 15 s) by trans-sided ATP hydrolysis, which triggers release of GroES. For the substrate protein rhodanese, only approximately 15% of cis-localized molecules attain their native form before hydrolysis. A major question concerning the GroEL mechanism is whether both native and non-native forms are released from the cis complex. Here we address this question using a 'cis-only' mixed-ring GroEL complex that binds polypeptide and GroES on only one of its two rings. This complex mediates refolding of rhodanese but, as with wild-type GroEL, renaturation is quenched by addition of mutant GroEL 'traps', which bind but do not release polypeptide substrate. This indicates that non-native forms are released from the cis complex. Quenching of refolding by traps was also observed under physiological conditions, both in undiluted Xenopus oocyte extract and in intact oocytes. We conclude that release of non-native forms from GroEL in vivo allows a kinetic partitioning among various chaperones and proteolytic components, which determines both the conformation and lifetime of a protein.

Although a CCTG expansion in the gene encoding the zinc knuckle protein CNBP causes a common form of muscular dystrophy, the function of both human CNBP and its putative budding yeast ortholog Gis2 remain poorly understood. Here we report the protein interactions of Gis2 and the subcellular locations of both Gis2 and CNBP. We found that Gis2 exhibits RNA-dependent interactions with two proteins involved in mRNA recognition, the poly(A) binding protein and the translation initiation factor eIF4G. We show that Gis2 is a component of two large RNA-protein granules, processing bodies and stress granules, which contain translationally repressed mRNAs. Consistent with a functional ortholog, CNBP also associates with the poly(A) binding protein and accumulates in stress granules during arsenite treatment of human cells. These results implicate both Gis2 and CNBP in mRNA handling during stress.

A role in folding newly translated cytoskeletal proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide 1 (TCP1). In this study, we investigated tubulin and actin biogenesis in Chinese hamster ovary (CHO) cells. When extracts of pulse-labeled cells were analyzed by anion-exchange and size-exclusion chromatography, newly synthesized alpha-tubulin, beta-tubulin, and actin were observed to enter a large molecular mass complex (approximately 900 kDa). These proteins were released from this complex capable, in the case of tubulin, of forming heterodimers. The large molecular mass complexes coeluted with TCP1 and could be immunoprecipitated by using an anti-TCP1 antibody. These findings demonstrate that there is a cytosolic pathway for folding tubulin and actin in vivo that involves the TCP1 complex.

A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking., Author Summary A new animal model of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) is presented. Two percent of ALS cases result from heritable mutations affecting the abundant enzyme superoxide dismutase (SOD1). Such mutations have been indicated to impair the folding and stability of the enzyme, leading it to misfold and aggregate in motor neurons, associated with the paralyzing disease. Here, when a mutant form of human SOD1 was produced in neurons of C. elegans worms, it led to a severe locomotor defect—the worms were essentially paralyzed. The protein formed aggregates in the neurons, including an intermediate form of aggregate, soluble oligomers, that has been linked to toxicity to cells. By contrast, worms expressing the normal version of human SOD1 protein exhibited normal movement and no aggregation. The movement defect was further analyzed using chemical inhibitors and found to result from defective function of synapses, the connections made between neurons, and between neurons and muscle. Finally, in a screen using RNA interference, we observed that the worms' aggregation and locomotor condition was worsened when a class of molecules called molecular chaperones, which assist protein folding in the cell, were impaired in function. This is consistent with the idea that misfolded SOD1 is directly involved with causing the neuronal dysfunction.

SummaryChaperonin action is controlled by cycles of nucleotide binding and hydrolysis. Here, we examine the effects of nucleotide binding on an archaeal group 2 chaperonin. In contrast to the ordered apo state of the group 1 chaperonin GroEL, the unliganded form of the homo-16-mer Methanococcus maripaludis group 2 chaperonin is very open and flexible, with intersubunit contacts only in the central double belt of equatorial domains. The intermediate and apical domains are free of contacts and deviate significantly from the overall 8-fold symmetry. Nucleotide binding results in three distinct, ordered 8-fold symmetric conformations—open, partially closed, and fully closed. The partially closed ring encloses a 40% larger volume than does the GroEL-GroES folding chamber, enabling it to encapsulate proteins up to 80 kDa, in contrast to the fully closed form, whose cavities are 20% smaller than those of the GroEL-GroES chamber.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.

Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable “lid” structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding?

SummaryThe cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding of substrate proteins bearing “tag” sequences, such as the 11-residue ssrA sequence appended to proteins translationally stalled at ribosomes. Unfolding is coupled to translocation through a central channel into the associating protease, ClpP. To explore the topology and mechanism of ClpA action, we carried out chemical crosslinking and functional studies. Whereas a tag from RepA protein crosslinked proximally to the flexible N domains, the ssrA sequence in GFP-ssrA crosslinked distally in the channel to a segment of the distal ATPase domain (D2). Single substitutions placed in this D2 loop, and also in two apparently cooperating proximal (D1) loops, abolished binding of ssrA substrates and unfolded proteins lacking tags and blocked unfolding of GFP-RepA. Additionally, a substitution adjoining the D2 loop allowed binding of ssrA proteins but impaired their translocation. This loop, as in homologous nucleic-acid translocases, may bind substrates proximally and, coupled with ATP hydrolysis, translocate them distally, exerting mechanical force that mediates unfolding.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.

Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL-GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL-GroES. Surprisingly, trans-only also folded smaller GroEL-GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a trans-only complex rescued a GroEL-deficient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL-GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more efficient folding.

The class II chaperonin CCT facilitates protein folding by a process that is not well-understood. One striking feature of this chaperonin is its apparent selectivity in vivo, folding only actin, tubulin, and several other proteins. In contrast, the class I chaperonin GroEL is thought to facilitate the folding of many proteins within Escherichia coli. It has been proposed that this apparent selectivity is associated with certain regions of a substrate protein's primary structure. Using limiting amounts of beta-tubulin, beta-tubulin mutants, and beta-tubulin/ftsZ chimeras, we assessed the contribution of select regions of beta-tubulin to CCT binding. In a complementary study, we investigated inter-ring communication in CCT where we exploited polypeptide binding sensitivity to nucleotide to quantitate nucleotide binding. beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K(D) approximately 3 nM; its apparent binding free energy, DeltaG, ca. -11.8 kcal/mol). Despite this, the interactions appear to be weak and distributed throughout much of the sequence, although certain sites ("hot spots") may interact somewhat more strongly with CCT. Globally averaged over the beta-tubulin sequence, these interactions appear to contribute ca. -9 to -11 cal/mol per residue, and to account for no more than 50-60% of the total binding free energy. We propose that a conformation change or deformation induced in CCT by substrate binding provides the missing free energy which stabilizes the binary complex. We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "intrinsic" affinities for its substrates. This strategy allows for dynamic interactions between chaperonin and bound substrate, which may facilitate folding on the interior surface of CCT in the absence of nucleotide and/or productive release of bound polypeptide into the central cavity upon subsequent MgATP binding. CCT displayed negative inter-ring cooperativity like GroEL. When ring 1 of CCT bound MgATP or beta-tubulin, the affinity of ring 2 for polypeptide or nucleotide was apparently reduced approximately 100-fold.

The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.

Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit.

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.

Summary The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber., Graphical Abstract Highlights ► Single-particle cryo-EM identifies seven ATP-bound structures of GroEL chaperonin ► Analysis of structures suggests trajectory of domain movements triggered by ATP binding ► The structures delineate a mechanism for mechanical unfolding of polypeptide substrate ► Model suggests an intermediate conformation binding substrate and GroES simultaneously, Cryo-EM analysis reveals a series of conformational steps through which GroEL binds polypeptide substrates, stretches and unfolds them, and then moves them into a folding chamber that promotes adoption of the native state.

The eukaryotic cytosolic chaperonin, CCT, plays an essential role in mediating ATP-dependent folding of actin and tubulin. There is debate about whether it mediates folding through a single round of association followed by release of native forms, or through cycles of binding and full release in which only a fraction of released molecules reaches native form in any cycle. We examine the fate of newly synthesized substrate proteins bound to CCT in reticulocyte lysate or intact Xenopus oocytes. When a chaperonin “trap,” able to bind but not release substrate protein, is introduced, production of the native state is strongly inhibited, associated with transfer to trap. While predominantly nonnative forms of actin, tubulin, and a newly identified substrate, Gα-transducin, are released from CCT, a small fraction reaches native form with each round of release, inaccessible to trap. This overall mechanism resembles that of the bacterial chaperonin, GroEL.

Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.

Sucrose hydrolysis has been shown to be catalyzed by the presence of imidazole at an acid pH. The active species was determined to be the positively charged imidazolium ion. The results are interpreted to offer a possible explanation of the first step of the dextransucrase-catalyzed formation of dextran from sucrose.