The interactions of molecular chaperones with client proteins: why are they so weak?

The major classes of molecular chaperones have highly variable sequences, sizes, and shapes, yet they all bind to unfolded proteins, limit their aggregation, and assist in their folding. Despite the central importance of this process to protein homeostasis, it has not been clear exactly how chaperones guide this process or whether the diverse families of chaperones use similar mechanisms. For the first time, recent advances in NMR spectroscopy have enabled detailed studies of how unfolded, “client” proteins interact with both ATP-dependent and ATP-independent classes of chaperones. Here, we review examples from four distinct chaperones, Spy, Trigger Factor, DnaK, and HscA-HscB, highlighting the similarities and differences between their mechanisms. One striking similarity is that the chaperones all bind weakly to their clients, such that the chaperone–client interactions are readily outcompeted by stronger, intra- and intermolecular contacts in the folded state. Thus, the relatively weak affinity of these interactions seems to provide directionality to the folding process. However, there are also key differences, especially in the details of how the chaperones release clients and how ATP cycling impacts that process. For example, Spy releases clients in a largely folded state, while clients seem to be unfolded upon release from Trigger Factor or DnaK. Together, these studies are beginning to uncover the similarities and differences in how chaperones use weak interactions to guide protein folding.     

Production,characterization, and assessment of a stable analog of the response regulator CheY‐phosphate from Thermotoga maritima

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY‐phosphate have been challenging due to the rapid hydrolysis of the aspartyl‐phosphate in vitro. An inert analog of Tm CheY‐phosphate, phosphono‐CheY, was synthesized by chemical modification and purified by cation‐exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono‐CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono‐CheY bound a peptide derived from FliM, a native partner of CheY‐phosphate, with a dissociation constant of ～29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono‐CheY is a useful and unique analog of Tm CheY‐phosphate.     

Blubber cortisol qualitatively reflects circulating cortisol concentrations in bottlenose dolphins

Stress hormones, released into circulation as a consequence of disturbance, are classically assayed from blood samples but may also be detected in a variety of matrices. Blubber and fecal samples can be remotely collected from free‐ranging cetaceans without the confounding hormone elevations associated with chase, capture, and handling required to collect blood samples. The relationship between cortisol concentrations in circulation with that of blubber and feces, however, is unknown. To assess these associations, we elevated cortisol by orally administering hydrocortisone for five days in five bottlenose dolphins. Voluntary blood and fecal samples were collected daily; blubber biopsies were collected on day one, just prior to hydrocortisone administration, and days three and five of hydrocortisone administration. We evaluated subsequent changes in several circulating stress hormones as well as cortisol and glucocorticoid metabolites in blubber and feces, respectively. There was a significant association between cortisol levels in serum and in blubber (F_1,12.7 = 14.3, P < 0.01, mR² = 0.57) despite substantial variability in blubber cortisol levels. Counterintuitively, fecal cortisol metabolite levels were inversely related to serum cortisol. The relationship between serum and blubber cortisol levels suggests blubber samples from remote sampling may be useful to detect stress loads in this species.     

Bacterial tail anchors can target to the mitochondrial outer membrane

Background

During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol.

Results

Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM.

Conclusions

Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

Reviewers

This article was reviewed by Michael Ryan and Thomas Simmen.

     

Characterization of thyroid hormone receptor alpha (TRalpha)-specific analogs with varying inner- and outer-ring substituents

Analogs of the TRalpha-specific thyromimetic CO23 were synthesized and analyzed in vitro using competitive binding and transactivation assays. Like CO23, all analogs bind to both thyroid hormone receptor subtypes with about the same affinity; however, modification of CO23 by derivatization of the 3' position of the outer-ring or replacement of the inner-ring iodides with bromides attenuates binding. Despite lacking a preference in binding to TRalpha, all analogs display TRalpha-specificity in transactivation assays using U2OS and HeLa cells. At best, several agonists exhibit an approximately 6-12-fold preference in transactivation when tested with TRalpha in HeLa cells. One analog, CO24, showed in vivo TRalpha-specific action in a tadpole metamorphosis assay.     

Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.     

Antiphonal call timing in marmosets is behaviorally significant: interactive playback experiments

Studies of primate vocal communication systems have generally focused on vocalizations and the information they convey to conspecifics. But the vocalizations are not the only sources of information. Aspects of each species vocal behaviors are likely to be communicatively rich as well. During vocal interactions, for example, the latency delay between the calls could communicate an important message to the signal receiver, such as an interest and willingness to socialize. Here we employed novel, interactive playback software to address this issue in the antiphonal calling behavior of common marmosets. In these experiments, we parametrically varied the latency delay of antiphonal call stimuli and measured its effects on subjects’ resultant vocal behavior. Results showed that marmosets produced successively fewer antiphonal call responses during test conditions with increasing latency delays. Moreover, although subjects produced significantly more antiphonal than spontaneous calls in conditions with antiphonal call timing delays up to 9 s, a longer delay resulted in a significant decline in calling. These data suggest that antiphonal call timing is a salient cue for maintaining antiphonal calling interactions and may be used by marmosets to determine whether a subsequent call is produced in response to or independently of their own.     

The effects of diet and caloric restriction on adipose tissue fatty acid signatures of tufted puffin (<Emphasis Type="Italic">Fratercula cirrhata</Emphasis>) nestlings

Fatty acid (FA) signature analysis is a powerful tool to investigate foraging ecology and food web dynamics in marine ecosystems. However, use of FA signatures to qualitatively or quantitatively infer diets is potentially complicated by effects of nutritional state on lipid metabolism. Estimation of diets using the quantitative fatty acid signature analysis (QFASA) model requires the use of calibration coefficients to account for predator metabolism of individual FAs. We conducted a captive feeding experiment to determine the effects of a 50% reduction in food intake on growth rate and adipose tissue FA signatures of tufted puffin (Fratercula cirrhata) nestlings, a species that routinely experiences food restriction during growth. FA signatures of chicks fed low- and high-calorie diets both exhibited a change in composition in response to the dietary shift with the direction of change in the composition of individual FAs matching the direction of change in the dietary FAs. Despite a growth rate in the restricted nestlings that was 38% of those in the well-fed group, rates of FA turnover were not different between high and low-calorie treatments, and turnover was close to, but not entirely complete, after 27 days on both high-calorie and restricted diets. FA signatures of tufted puffin nestlings were significantly affected by caloric restriction, but these effects were much less pronounced than those of dietary turnover, and calibration coefficients of puffins fed low and high-calorie diets were highly correlated. Our results demonstrate that changes in physiological state can affect FA metabolism, but future research is required to better understand whether the size of these effects is sufficient to substantially alter diet estimation using the QFASA model.     

Ab initio maximum likelihood reconstruction from cryo electron microscopy images of an infectious virion of the tailed bacteriophage P22 and maximum likelihood versions of Fourier Shell Correlation appropriate for measuring resolution of spherical or cylindrical objects

A maximum likelihood reconstruction method for an asymmetric reconstruction of the infectious P22 bacteriophage virion is described and demonstrated on a subset of the images used in [Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A., Potter, C.S., Carragher, B., Johnson, J.E., 2006. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312(5781), 1791–1795]. The method makes no assumptions at any stage regarding the structure of the phage tail or the relative rotational orientation of the phage tail and capsid but rather the structure and the rotation angle are determined as a part of the analysis. A statistical method for determining resolution consistent with maximum likelihood principles based on ideas for cylinders analogous to the ideas for spheres that are embedded in the Fourier Shell Correlation method is described and demonstrated on the P22 reconstruction. With a correlation threshold of .95, the resolution in the tail measured radially is greater than (33.3 Å) and measured axially is greater than (70.6 Å) both with probability p=0.02.     

Intact fetal ovarian cord formation promotes mouse oocyte survival and development

Background  

Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined.