Parallel chromatography and in situ scattering to interrogate competing protein aggregation pathways.

Protein aggregation can follow different pathways, and these can result in different net aggregation rates and kinetic profiles. α-chymotypsinogen A (aCgn) was used as a model system to quantitatively and qualitatively assess an approach that combines ex situ size-exclusion chromatography (SEC) with in situ laser scattering (LS) to monitor aggregation vs. time. Aggregation was monitored for a series of temperatures and initial dimer (ID) levels for starting conditions that were primarily (> 97%) monomer, and under initial-rate conditions (limited to low monomer conversion-less than 20% monomer mass loss), as these conditions are of most to interest to many pharmaceutical and biotechnology applications. SEC results show that modest decreases of ID levels can greatly reduce monomer loss rates, but do not affect the effective activation energy for aggregation. The normalized aggregation rates determined from LS were typically ∼ 1 order of magnitude higher than the corresponding rates from SEC. Furthermore, LS signals vs. time became variable and highly nonlinear with decreasing ID level, temperature, and/or total protein concentration. Temperature-cycling LS experiments showed this corresponded to conditions where dimer/oligomer "seeding" was suppressed, and high levels of reversible oligomers ("prenuclei") were formed prior to "nucleation" and growth of stable aggregates. In those conditions, aggregation rates inferred from LS and SEC are greatly different, as the techniques monitor different stages of the aggregation process. Overall, the results illustrate an approach for interrogating non-native protein aggregation pathways, and potential pitfalls if one relies on a single method to monitor aggregation-this holds more generally than the particular methods here.

Oral midodrine treatment accelerates the liberation of intensive care unit patients from intravenous vasopressor infusions.

PURPOSE: Persistent low-level hypotension represents a barrier to discharging patients from the intensive care unit (ICU). Midodrine may be an effective adjunct to wean intravenous (IV) vasopressors and permit ICU discharge. We tested the hypothesis that midodrine, given to patients on IV vasopressors who otherwise met ICU discharge criteria, increased the magnitude of change in IV vasopressor rate. MATERIALS AND METHODS: This was a prospective, observational study in 20 adult surgical ICU patients who met ICU discharge criteria except for an IV vasopressor requirement. We compared the change in phenylephrine equivalent rates during the day before midodrine to the change in phenylephrine equivalent rates after midodrine initiation and analyzed changes in total body fluid balance, heart rate, mean arterial pressure, and white blood cell count during this period. RESULTS: Patients received 41.0±33.4 µg/min of phenylephrine equivalents and the change in IV vasopressor rate (slope) decreased significantly from -0.62 µg/min per hour of phenylephrine equivalents before midodrine to -2.20 µg/min per hour following the initiation of midodrine treatment (P=.012). Change in total body fluid balance, heart rate, mean arterial pressure, and white blood cell count did not correlate with change in IV vasopressor rate. CONCLUSION: Midodrine treatment was associated with an increase in the magnitude of decline of the IV vasopressor rate. Oral midodrine may facilitate liberation of surgical ICU patients from an IV vasopressor infusion, and this may affect discharge readiness of patients from the ICU.

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance.

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.

An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP).

The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins.