Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

The Hsp90 chaperone is a central node of protein homeostasis activating a large number of diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies define distinct conformational states of the mechanistic core implying structural changes that have not yet been observed in solution. Here, we engineered one-nanometer fluorescence probes based on photo-induced electron transfer into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement are mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilizes the lid of apo Hsp90, suggesting an early role in the catalytic cycle

Bolaamphiphile analogues of 12-bis-THA Cl2 are potent antimicrobial therapeutics with distinct mechanisms of action against bacterial, mycobacterial, and fungal pathogens.

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used

Regulation of activation-induced deaminase stability and antibody gene diversification by Hsp90

Hsp90 stabilizes and prevents degradation of cytoplasmic activation-induced deaminase

Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1

Complex conformational dynamics are essential for function of the dimeric molecular cha- perone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimer- ization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90

Dissolving the Dichotomies Between Online and Campus-Based Teaching: a Collective Response to The Manifesto for Teaching Online (Bayne et al. 2020)

This article is a collective response to the 2020 iteration of The Manifesto for Teaching Online. Originally published in 2011 as 20 simple but provocative statements, the aim was, and continues to be, to critically challenge the normalization of education as techno-corporate enterprise and the failure to properly account for digital methods in teaching in Higher Education. The 2020 Manifesto continues in the same critically provocative fashion, and, as the response collected here demonstrates, its publication could not be timelier. Though the Manifesto was written before the Covid-19 pandemic, many of the responses gathered here inevitably reflect on the experiences of moving to digital, distant, online teaching under unprecedented conditions. As these contributions reveal, the challenges were many and varied, ranging from the positive, breakthrough opportunities that digital learning offered to many students, including the disabled, to the problematic, such as poor digital networks and access, and simple digital poverty. Regardless of the nature of each response, taken together, what they show is that The Manifesto for Teaching Online offers welcome insights into and practical advice on how to teach online, and creatively confront the supremacy of face-to-face teaching

UCS protein function is partially restored in the Saccharomyces cerevisiae she4 mutant with expression of the human UNC45-GC, but not UNC45-SM

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90β isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90β. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90β in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved-yeast to man-in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking-events also facilitated by the myosins in yeast

Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90

Hsp90 is an essential eukaryotic chaperone with a role in folding specific “client” proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions

ADAM8 signaling drives neutrophil migration and ARDS severity

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain

Downregulation of the Hsp90 System Causes Defects in Muscle Cells of Caenorhabditis Elegans

The ATP-dependent molecular chaperone Hsp90 is required for the activation of a variety of client proteins involved in various cellular processes. Despite the abundance of known client proteins, functions of Hsp90 in the organismal context are not fully explored. In Caenorhabditis elegans, Hsp90 (DAF-21) has been implicated in the regulation of the stress-resistant dauer state, in chemosensing and in gonad formation. In a C. elegans strain carrying a DAF-21 mutation with a lower ATP turnover, we observed motility defects. Similarly, a reduction of DAF-21 levels in wild type nematodes leads to reduced motility and induction of the muscular stress response. Furthermore, aggregates of the myosin MYO-3 are visible in muscle cells, if DAF-21 is depleted, implying a role of Hsp90 in the maintenance of muscle cell functionality. Similar defects can also be observed upon knockdown of the Hsp90-cochaperone UNC-45. In life nematodes YFP-DAF-21 localizes to the I-band and the M-line of the muscular ultrastructure, but the protein is not stably attached there. The Hsp90-cofactor UNC-45-CFP contrarily can be found in all bands of the nematode muscle ultrastructure and stably associates with the UNC-54 containing A-band. Thus, despite the physical interaction between DAF-21 and UNC-45, apparently the two proteins are not always localized to the same muscular structures. While UNC-45 can stably bind to myofilaments in the muscular ultrastructure, Hsp90 (DAF-21) appears to participate in the maintenance of muscle structures as a transiently associated diffusible factor