SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from glutamine-contributing SNAREs (Q-SNAREs) embedded in one membrane and an ...arginine-contributing SNARE (R-SNARE) embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp colocalizes with and binds to VAMP7, an R-SNARE that is involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7-SNARE motif is trapped between Varp and the VAMP7 longin domain, and hence Varp kinetically inhibits the ability of VAMP7 to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as VAMP7, such as Rab32-GTP.
VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show ...that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.
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► AP3-containing vesicles transport VAMP7 and LAMP1 from early to late endosomes ► The ear/trunk linker of the AP3 δ-subunit binds in a groove on a VAMP7 longin domain ► Binding of VAMP by AP3 can only occur when VAMP is part of a cis-SNARE complex ► VAMP7 is not required for the fusion of AP3-containing vesicles with late endosomes
VAMP7 SNARE complexes on late endosomes determine the specificity of membrane fusion there. But how is VAMP7 localization maintained? Kent et al. define the structure of and requirement for VAMP7 interactions with the cargo adaptor AP3. AP3 uses distinct surfaces for VAMP7 versus other cargoes to recycle postfusion cis-SNARE complexes.
Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active ...humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.
The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V1Vo ATPases and with F1Fo ...ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V1Vo ATPases. The chimeric nature of the A1Ao ATP synthase from the archaeon Methanosarcina mazei Gö1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5 A resolution, providing the first view of the so-called non-catalytic subunit of the A1Ao ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic alpha subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A1Ao ATP synthase is presented.
Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes, and biochemical properties. Here, we report a fully ...active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site-1 and site-2 interactions.
The A
1A
O ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V
1V
O ATPases and with F
...1F
O ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V
1V
O ATPases. The chimeric nature of the A
1A
O ATP synthase from the archaeon
Methanosarcina mazei Gö1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5
Å resolution, providing the first view of the so-called non-catalytic subunit of the A
1A
O ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic α subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A
1A
O ATP synthase is presented.
On the basis of current treatment guidelines, we developed and validated a medication-based chronic disease score (medCDS) and tested its association with all-cause mortality of older outpatients.
...Considering the most prevalent chronic diseases in the elderly German population, we compiled a list of evidence-based medicines used to treat these disorders. Based on this list, a score (medCDS) was developed to predict mortality using data of a large longitudinal cohort of older outpatients (training sample; MultiCare Cohort Study). By assessing receiver-operating characteristics (ROC) curves, the performance of medCDS was then confirmed in independent cohorts (ESTHER, KORA-Age) of community-dwelling older patients and compared with already existing medication-based scores and a score using selected anatomical-therapeutic-chemical (ATC) codes.
The final medCDS score had an ROC area under the curve (AUC) of 0.73 (95% CI 0.70-0.76). In the validation cohorts, its ROC AUCs were 0.79 (0.76-0.82, KORA-Age) and 0.74 (0.71-0.78, ESTHER), which were superior to already existing medication-based scores (RxRisk, CDS) and scores based on pharmacological ATC code subgroups (ATC3) or age and sex alone (Age&Sex).
A new medCDS, which is based on actual treatment standards, predicts mortality of older outpatients significantly better than already existing scores.
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