To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by ...acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation.
Summary
The catalytic
A
1 subunit of cholera toxin (
CTA1
) has a disordered structure at 37°C. An interaction with host factors must therefore place
CTA1
in a folded conformation for the ...modification of its
Gs
α target which resides in a lipid raft environment. Host
ADP
‐ribosylation factors (
ARFs
) act as
in vitro
allosteric activators of
CTA1
, but the molecular events of this process are not fully characterized. Isotope‐edited
F
ourier transform infrared spectroscopy monitored
ARF6
‐induced structural changes to
CTA1
, which were correlated to changes in
CTA1
activity. We found
ARF6
prevents the thermal disordering of structured
CTA1
and stimulates the activity of stabilized
CTA1
over a range of temperatures. Yet
ARF6
alone did not promote the refolding of disordered
CTA1
to an active state. Instead, lipid rafts shifted disordered
CTA1
to a folded conformation with a basal level of activity that could be further stimulated by
ARF6
. Thus,
ARF
alone is unable to activate disordered
CTA1
at physiological temperature: additional host factors such as lipid rafts place
CTA1
in the folded conformation required for its
ARF
‐mediated activation. Interaction with
ARF
is required for
in vivo
toxin activity, as enzymatically active
CTA1
mutants that cannot be further stimulated by
ARF6
fail to intoxicate cultured cells.
Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. ...Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.
Human papillomavirus (HPV) infection has been causally linked to six cancers, and many disproportionately affect minorties. This study reports on the development and effectiveness of an intervention ...aimed at increasing HPV vaccine uptake among African American and Hispanic pediatric patients in safety-net clinics.
Formative research, community engagement, and theory guided development of the intervention. A clustered, non-randomized controlled pragmatic trial was conducted in four clinics providing healthcare for the underserved in Tennessee, U.S., with two intervention sites and two usual care sites. Patients aged 9-18 years (N = 408) and their mothers (N = 305) enrolled, with children clustered within families. The intervention consisted of two provider/staff training sessions and provision of patient education materials, consisting of a video/flyer promoting HPV vaccine. Medical records were reviewed before/after the initial visit and after 12 months.
At the initial visit, provision of patient education materials and provider recommendation were higher at intervention sites versus usual care sites, and receipt of HPV vaccine was higher at intervention sites (45.4% versus 32.9%) but not significantly after adjusting for patient's age and mother's education. Provider recommendation, but not education materials, increased the likelihood of vaccine receipt at the initial visit, although over one-third of intervention mothers cited the flyer/video as motivating vaccination. Completion of the 3-dose series at follow-up was lower in the intervention arm.
Future interventions should combine patient education, intensive provider/staff education, and patient reminders. Research should compare patient education focusing on HPV vaccine only versus all adolescent vaccines.
Retrospectively registered with ClinicalTrials.gov NCT02808832 , 9/12/16.
This study examined demographic and lifestyle factors that influenced decisions and obstacles to being screened for colorectal cancer in low-income African Americans in three urban Tennessee cities. ...As part of the Meharry Community Networks Program (CNP) needs assessment, a 123-item community survey was administered to assess demographic characteristics, health care access and utilization, and screening practices for various cancers in low-income African Americans. For this study, only African Americans 50 years and older (n = 460) were selected from the Meharry CNP community survey database. There were several predictors of colorectal cancer screening such as being married and having health insurance (
P
< .05). Additionally, there were associations between obstacles to screening and geographic region such as transportation and health insurance (
P
< .05). Educational interventions aimed at improving colorectal cancer knowledge and screening rates should incorporate information about obstacles and predictors to screening.
This study examined demographic and lifestyle factors that influenced decisions to get screened for prostate cancer in low-income African Americans in three urban Tennessee cities. It also examined ...obstacles to getting screened. As part of the Meharry Community Networks Program (CNP) needs assessment, a 123-item community survey was administered to assess demographic characteristics, health care access and utilization, and screening practices for various cancers in low-income African Americans. For this study, only African American men 45 years and older (n=293) were selected from the Meharry CNP community survey database. Participants from Nashville, those who were older, obese, and who had health insurance were more likely to have been screened (p<.05). Additionally, there were associations between obstacles to screening (such as cost and transportation) and geographic region (p<.05). Educational interventions aimed at improving prostate cancer knowledge and screening rates should incorporate information about obstacles to and predictors of screening.
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