Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin.

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration.

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects.

Double mutant MBP refolds at same rate in free solution as inside the GroEL/GroES chaperonin chamber when aggregation in free solution is prevented.

Under "permissive" conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5-10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering.

A biophysical glance at the outer surface of the membrane transporter SGLT1.

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein.

ATP-triggered ADP release from the asymmetric chaperonin GroEL/GroES/ADP7 is not the rate-limiting step of the GroEL/GroES reaction cycle.

The GroEL/GroES protein folding chamber is formed and dissociated by ATP binding and hydrolysis. ATP hydrolysis in the GroES-bound (cis) ring gates entry of ATP into the opposite unoccupied trans ring, which allosterically ejects cis ligands. While earlier studies suggested that hydrolysis of cis ATP is the rate-limiting step of the cycle (t1/2 approximately 10 s), a recent study suggested that ADP release from the cis ring may be rate-limiting (t1/2 approximately 15-20 s).

GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state.

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide.

D-Glucose-recognition and phlorizin-binding sites in human sodium/D-glucose cotransporter 1 (hSGLT1): a tryptophan scanning study.

In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.

A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains.

In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate.

Sodium-independent low-affinity D-glucose transport by human sodium/D-glucose cotransporter 1: critical role of tryptophan 561.

Although there is no evidence of significant Na-independent glucose flux in tissues naturally expressing SGLT1, previous kinetic and biophysical studies suggest that sodium/d-glucose cotransporter 1 (hSGLT1) can facilitate sodium-independent d-glucose transport and may contain more than one sugar binding site. In this work, we analyze the kinetic properties and conformational states of isolated hSGLT1 reconstituted in liposomes by transport and fluorescence studies in the absence of sodium. In the transport studies with hSGLT1, significant sodium-independent phlorizin inhibitable alpha-methyl d-glucopyranoside (alpha-MDG) uptake was observed which amounted to approximately 20% of the uptake observed in the presence of a sodium gradient.

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/D-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence.

Recombinant purified human sodium/D-glucose cotransporter1 (hSGLT1) was reconstituted in a functional form into phospholipid vesicles and its conformational states in the absence and presence of ligands and inhibitors were probed by intrinsic tryptophan fluorescence. In the presence of sodium, sugars increase intrinsic fluorescence (maximum 17%) in a saturable manner in the following order alpha-MDG >D-Glu approximately D-Gal >> D-Man >D-All, with no effect of L-Glu. Apparent affinities ranging from 0.

High-yield functional expression of human sodium/d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced conformational changes as studied by tryptophan fluorescence.

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P.

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein.

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths.

Phlorizin recognition in a C-terminal fragment of SGLT1 studied by tryptophan scanning and affinity labeling.

SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2'-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by site-directed single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564-638) were expressed in Escherichia coli and purified to homogeneity.