Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action
. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of ...substances from the cytosol
and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance
. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.
Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for ...antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.
Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to ...identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no β5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)–1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA–mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bor-tezomib. Importantly, OSI-906 in combination with bortezomib also overcame bor-tezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.
Aims
Bulking agents are a minimally invasive treatment option for women with stress urinary incontinence (SUI) or stress‐predominant mixed urinary incontinence (MUI). The aim of this study was to ...evaluate long‐term efficacy and safety following treatment with Bulkamid as a primary procedure for SUI or stress‐predominant MUI.
Methods
This was an Institutional Review Board‐approved single‐center retrospective study of female patients with SUI or stress‐predominant MUI who had undergone injection with Bulkamid since 2005 and had completed 7 years of follow up. The primary endpoint was patient satisfaction measured on a four‐point scale as cured, improved, unchanged, or worse. Secondary outcomes included the number of incontinence pads used, International Consultation on Incontinence Questionnaire‐Short Form (ICIQ‐UI SF) scores, Visual Analog Scale Quality of Life (VAS QoL), reinjection rates, and perioperative and postoperative complications.
Results
A total of 1,200 patients were treated with Bulkamid since 2005 and of these, 388 (32.3%) had completed 7 years of follow‐up. A total of 67.1% of the patients reported feeling cured or improved if Bulkamid was a primary procedure, 11.1% reported no change, and 2.3% reported worsening of incontinence. A total of 19.5% of patients received a subsequent other incontinence procedure. The ICIQ‐UI SF was reduced by 8.6 points. VAS QoL improved by a mean of 4.3 points. Postoperative complications were transient. Prolonged bladder emptying time was reported in 15.3% of patients and urinary tract infection in 3.5%.
Conclusions
Bulkamid injections are an effective and safe first‐line treatment option for women with SUI or stress‐predominant MUI providing durable outcomes at 7 years.
Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among ...others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.
Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of ...expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.
Ions greatly influence protein structure–function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels ...near active calcium channels at the SR membrane and the impact of localized high calcium on the structure–function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure–function, establish a novel approach to understand the enthalpy of protein–ion interactions and the accompanying structural changes that may occur within the protein molecule.
Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term ...exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/β-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased β-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of β-catenin protein by up to 20-fold or more. This produced Wnt/β-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the β-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/β phosphorylation. Stimulation of Wnt/β-catenin signaling with recombinant Wnt-3a, or by overexpression of β-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of β-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/β-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.
The panel focused largely on the management of this complex disease and derived prudent, practical, and contemporary treatment strategies for the many subgroups of patients comprising the broad HCM ...disease spectrum. Because of the relatively low prevalence of HCM in general cardiologic practice (50), its diverse presentation, and mechanisms of death and disability and skewed patterns of patient referral (7,11,13,36-38,42,51-59), the level of evidence governing management decisions for drugs or devices has often been derived from non-randomized and retrospective investigations. Large-scale controlled and randomized study designs, such as those that have provided important answers regarding the management of coronary artery disease (CAD) and congestive heart failure (60-62), have generally not been available in HCM as a result of these factors. ...treatment strategies have necessarily evolved based on available data that have frequently been observational in design, sometimes obtained in relatively small patient groups, or derived from the accumulated clinical experience of individual investigators, and reasonable inferences drawn from other cardiac diseases.
The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is ...presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.
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