Structural transitions modulate the chaperone activities of Grp94.

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.

Insight into the Nucleotide Based Modulation of the Grp94 Molecular Chaperone Using Multiscale Dynamics.

Grp94, an ER-localized molecular chaperone, is required for the folding and activation of many membrane and secretory proteins. Client activation by Grp94 is mediated by nucleotide and conformational changes. In this work, we aim to understand how microscopic changes from nucleotide hydrolysis can potentiate large-scale conformational changes of Grp94. We performed all-atom molecular dynamics simulations on the ATP-hydrolysis competent state of the Grp94 dimer in four different nucleotide bound states. We found that Grp94 was the most rigid when ATP was bound. ATP hydrolysis or nucleotide removal enhanced mobility of the N-terminal domain and ATP lid, resulting in suppression of interdomain communication. In an asymmetric conformation with one hydrolyzed nucleotide, we identified a more compact state, similar to experimental observations. We also identified a potential regulatory role of the flexible linker, as it formed electrostatic interactions with the Grp94 M-domain helix near the region where BiP is known to bind. These studies were complemented with normal-mode analysis of an elastic network model to investigate Grp94's large-scale conformational changes. SPM analysis identified residues that are important in signaling conformational change, many of which have known functional relevance in ATP coordination and catalysis, client binding, and BiP binding. Our findings suggest that ATP hydrolysis in Grp94 alters allosteric wiring and facilitates conformational changes.

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress.

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.

Vulvar ulcer causing osteomyelitis of the pubic bone.

BACKGROUND: Osteomyelitis of the pubic bone is a rare entity. Risk factors for infection of the symphysis pubis and osteomyelitis of the pubic bone include direct trauma, previous urogynecologic procedures, extreme physical exercise, and immunocompromised state. The treatment modalities range from conservative antibiotic treatment to extensive surgery. CASE: A 49-year-old woman with multiple sclerosis and borderline diabetes mellitus presented with bloody vulvovaginal discharge. The source was found out to be an ulcer located above the urethra with exposure of the underlying symphysis pubis. Intraoperative debridement of the ulcer followed by bone biopsies demonstrated osteomyelitis of the pubic bone. Prolonged intravenous antibiotics and 4 operative debridements were needed before the osteomyelitis was adequately addressed and the defect could be closed with a bulbocavernosus flap. CONCLUSIONS: This is the first report of a severe case of osteomyelitis of the pubic bone arising from a vulvar ulcer.

A prospective study of the efficiency of the "code 333" process at the Ottawa hospital.

OBJECTIVE: This study evaluated the efficiency of the "Code 333" process at The Ottawa Hospital (TOH) during obstetric emergencies. A Code 333 is an overhead call made in TOH obstetrics units to activate an emergency response system. The code calls for resuscitative measures on a mother and/or a fetus and expedited delivery of a fetus considered at high risk of demise. Internationally, the recommended maximum time between the decision to deliver and actual delivery of an infant, referred to as decision-to-delivery interval (DDI), is 30 minutes. METHODS: The study was conducted over an 11-month period from February 2007 to January 2008 at The Ottawa Hospital Birthing Units (TOHBU)-Civic and General campuses. Data were collected during the code using validated documentation sheets. The day and time of decision, the indication and outcome of the code, the mode of anaesthesia used, and the condition of the baby at birth were recorded for time interval calculations. RESULTS: The median DDI for 85 emergency Caesarean sections was 16 minutes. Ninety-eight percent of TOH deliveries were completed within the recommended 30 minutes. Over one third of these deliveries were completed within 15 minutes. Urgent codes had a median DDI of 13 minutes, compared with 20 minutes for less urgent codes. Time of day or day of the week did not have any effect on DDI. CONCLUSION: The recommended DDI of 30 minutes was routinely achieved at TOHBU. DDI was prolonged in only 2% of codes during the study period, with no adverse outcome.

Vulvar edema.

Vulvar edema is associated with a variety of conditions. The edema can result from inflammatory conditions, infections, infestations, trauma, pregnancy, tumors and iatrogenic causes. At times, it is difficult to determine the cause of the vulvar edema. Treatment consists of determining the origin of the edema and giving the appropriate therapy for that diagnosis as well as the use of compression and, at times, lymphatic massage.

Management of vulvovaginal strictures/shortened vagina.

Vaginal strictures are generally difficult to manage and tend to reccur despite appropriate initial therapy. Vaginal dilation with or without surgery is the main stay of treatment. Causes, diagnosis and management of the vulvovaginal strictures are presented.