Probing the interface in a human co-chaperonin heptamer: residues disrupting oligomeric unfolded state identified

BACKGROUND: The co-chaperonin protein 10 (cpn10) assists cpn60 in the folding of nonnative polypeptides in a wide range of organisms. All known cpn10 molecules are heptamers of seven identical subunits that are linked together by β-strand interactions at a large and flexible interface. Unfolding of human mitochondrial cpn10 in urea results in an unfolded heptameric state whereas GuHCl additions result in unfolded monomers. To address the role of specific interface residues in the assembly of cpn10 we prepared two point-mutated variants, in each case removing a hydrophobic residue positioned at the subunit-subunit interface. RESULTS: Replacing valine-100 with a glycine (Val100Gly cpn10) results in a wild-type-like protein with seven-fold symmetry although the thermodynamic stability is decreased and the unfolding processes in urea and GuHCl both result in unfolded monomers. In sharp contrast, replacing phenylalanine-8 with a glycine (Phe8Gly cpn10) results in a protein that has lost the ability to assemble. Instead, this protein exists mostly as unfolded monomers. CONCLUSIONS: We conclude that valine-100 is a residue important to adopt an oligomeric unfolded state but it does not affect the ability to assemble in the folded state. In contrast, phenylalanine-8 is required for both heptamer assembly and monomer folding and therefore this mutation results in unfolded monomers at physiological conditions. Despite the plasticity and large size of the cpn10 interface, our observations show that isolated interface residues can be crucial for both the retention of a heptameric unfolded structure and for subunit folding

Conserved Sequence Repeats of IQGAP1 Mediate Binding to Ezrin

Mammalian IQGAP proteins all feature multiple ∼50 amino acid sequence repeats near their N-termini, and little is known about the function of these “Repeats”. We have expressed and purified the Repeats from human IQGAP1 to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin, and moesin. ERM proteins have an N-terminal FERM domain (4.1, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2) and a C-terminal actin-binding domain and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain, while no binding is seen for full-length “autoinhibited” ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains for signaling purposes

α2B-Adrenergic Receptor Interaction with Tubulin Controls Its Transport from the Endoplasmic Reticulum to the Cell Surface*

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α2B-adrenergic receptor (α2B-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α2B-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α2B-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules

Multi-dimensional data transmission using inverse-designed silicon photonics and microcombs.

The use of optical interconnects has burgeoned as a promising technology that can address the limits of data transfer for future high-performance silicon chips. Recent pushes to enhance optical communication have focused on developing wavelength-division multiplexing technology, and new dimensions of data transfer will be paramount to fulfill the ever-growing need for speed. Here we demonstrate an integrated multi-dimensional communication scheme that combines wavelength- and mode- multiplexing on a silicon photonic circuit. Using foundry-compatible photonic inverse design and spectrally flattened microcombs, we demonstrate a 1.12-Tb/s natively error-free data transmission throughout a silicon nanophotonic waveguide. Furthermore, we implement inverse-designed surface-normal couplers to enable multimode optical transmission between separate silicon chips throughout a multimode-matched fibre. All the inverse-designed devices comply with the process design rules for standard silicon photonic foundries. Our approach is inherently scalable to a multiplicative enhancement over the state of the art silicon photonic transmitters

Outcomes after ultramassive transfusion in the modern era: An Eastern Association for the Surgery of Trauma multicenter study

BACKGROUND Despite the widespread institution of modern massive transfusion protocols with balanced blood product ratios, survival for patients with traumatic hemorrhage receiving ultramassive transfusion (UMT) (defined as >= 20 U of packed red blood cells [RBCs]) in 24 hours) remains low and resource consumption remains high. Therefore, we aimed to identify factors associated with mortality in trauma patients receiving UMT in the modern resuscitation era. METHODS An Eastern Association for the Surgery of Trauma multicenter retrospective study of 461 trauma patients from 17 trauma centers who received >= 20 U of RBCs in 24 hours was performed (2014-2019). Multivariable logistic regression and Classification and Regression Tree analysis were used to identify clinical characteristics associated with mortality. RESULTS The 461 patients were young (median age, 35 years), male (82%), severely injured (median Injury Severity Score, 33), in shock (median shock index, 1.2; base excess, -9), and transfused a median of 29 U of RBCs, 22 U of fresh frozen plasma (FFP), and 24 U of platelets (PLT). Mortality was 46% at 24 hours and 65% at discharge. Transfusion of RBC/FFP >= 1.5:1 or RBC/PLT >= 1.5:1 was significantly associated with mortality, most pronounced for the 18% of patients who received both RBC/PLT and RBC/FFP >= 1.5:1 (odds ratios, 3.11 and 2.81 for mortality at 24 hours and discharge; both p = 1.5:1, with increased associated mortality. Maintaining focus on balanced ratios during UMT is critical, and consideration of advanced age, poor initial mental status, thrombocytopenia, and resuscitative thoracotomy can aid in prognostication