Telehealth during COVID-19 restrictions in patients with cardiovascular disease: impact on medication prescriptions and patient satisfaction.

BACKGROUND AND AIMS: Telehealth plays an integral part in healthcare delivery. The impact of telehealth and the COVID-19 pandemic on medication prescribing and patient satisfaction with telehealth in cardiology clinics remains unknown. METHODS: A retrospective study of cardiology clinic patients at an Australian tertiary hospital was conducted; 630 patients seen before the COVID-19 pandemic (0.6% telehealth) and 678 during the pandemic (91.2% telehealth) were included. Medication changes, new prescriptions and time to obtaining prescriptions after clinic were compared. To evaluate patients' experiences, cardiology clinic patients reviewed during the pandemic were prospectively invited to participate in an electronic survey sent to their mobile phones. RESULTS: The overall rates of medication changes made in the clinic between the prepandemic and the pandemic periods did not differ significantly (26.9% vs 25.8%). Compared with prepandemic, new cardiac medication prescriptions during clinic were significantly less (9.3% vs 2.5%; P < 0.0001) and recommendations to general practitioners (GP) to initiate cardiac medications were significantly more (2.6% vs 9.1%; P < 0.0001). Time to obtaining new prescriptions was significantly longer in the pandemic cohort (median 0 days (range: 0-32) vs 10.5 days (range: 0-231); P < 0.0001). Two hundred forty-three (32.7%) patients participated in the survey; 50% reported that telehealth was at least as good as face-to-face consultations. Most patients (61.5%) were satisfied with telehealth and most (62.9%) wished to see telehealth continued postpandemic. CONCLUSION: Telehealth during the COVID-19 pandemic was associated with greater reliance on GP to prescribe cardiac medications and delays in obtaining prescriptions among cardiology clinic patients. Although most patients were satisfied with telehealth services, nearly half of the cardiac patients expressed preference towards traditional face-to-face consultations.

Vector genome loss and epigenetic modifications mediate decline in transgene expression of AAV5 vectors produced in mammalian and insect cells.

Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (∼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.

Weighing the DNA Content of Adeno-Associated Virus Vectors with Zeptogram Precision Using Nanomechanical Resonators.

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

A novel chemically modified analogue of xenin-25 exhibits improved glucose-lowering and insulin-releasing properties.

BACKGROUND: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is rapidly degraded following release into the circulation. We hypothesized that substitution of all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme resistance and enhanced biological efficacy. METHODS: Peptide stability was assessed using murine plasma, in vitro insulin-releasing actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic parameters and pancreatic histology were assessed in high fat fed mice which had received saline vehicle or xenin-25(gln) twice-daily for 21 days. RESULTS: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high fat fed mice did not affect food intake, body weight or circulating insulin concentrations but significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and glucose-mediated insulin release. CONCLUSIONS: Xenin-25(gln) is resistant to plasma-mediated degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob mice. GENERAL SIGNIFICANCE: Glutamine (gln)-modified analogues of xenin may represent an attractive therapeutic approach for type 2 diabetes.

Safety, Stability and Pharmacokinetic Properties of (super)Factor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding.

PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 µg/mL), (super)FVa did not (6.7 µg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 µg/mL) or saline (5.6 µg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gß5-RGS7 complex.

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gß5-RGS7, the permanently associated heterodimer of G protein ß-subunit Gß5 and RGS7, a regulator of G protein signaling. Gß5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gß5-RGS7. In addition to the i3 loop, interaction of M3R with Gß5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gß5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gß5-RGS7 requires helix 8 and the central portion of the i3 loop.

Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch.

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1-WW2 tandem module of WW domain-containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1-WW2 tandem module to an array of putative proline-proline-x-tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1-WW2 tandem module of WWOX.

Effect of osmolytes on the binding of EGR1 transcription factor to DNA.

Osmolytes play a key role in maintaining protein stability and mediating macromolecular interactions within the intracellular environment of the cell. Herein, we show that osmolytes such as glycerol, sucrose, and polyethylene glycol 400 (PEG400) mitigate the binding of early growth response (protein) 1 (EGR1) transcription factor to DNA in a differential manner. Thus, while physiological concentrations of glycerol only moderately reduce the binding affinity, addition of sucrose and PEG400 is concomitant with a loss in the binding affinity by an order of magnitude. This salient observation suggests that EGR1 is most likely subject to conformational equilibrium and that the osmolytes exert their effect via favorable interactions with the unliganded conformation. Consistent with this notion, our analysis reveals that while EGR1 displays rather high structural stability in complex with DNA, the unliganded conformation becomes significantly destabilized in solution. In particular, while liganded EGR1 adopts a well-defined arc-like architecture, the unliganded protein samples a comparatively large conformational space between two distinct states that periodically interconvert between an elongated rod-like shape and an arc-like conformation on a submicrosecond time scale. Consequently, the ability of osmolytes to favorably interact with the unliganded conformation so as to stabilize it could account for the negative effect of osmotic stress on EGR1-DNA interaction observed here. Taken together, our study sheds new light on the role of osmolytes in modulating a key protein-DNA interaction.

Vascular remodeling underlies rebleeding in hemophilic arthropathy.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

A novel glucagon-like peptide-1 (GLP-1)/glucagon hybrid peptide with triple-acting agonist activity at glucose-dependent insulinotropic polypeptide, GLP-1, and glucagon receptors and therapeutic potential in high fat-fed mice.

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA(2)]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA(2)]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA(2)]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA(2)]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA(2)]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA(2)]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA(2)]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.

Enthalpic factors override the polyelectrolyte effect in the binding of EGR1 transcription factor to DNA.

Protein-DNA interactions are highly dependent upon salt such that the binding affinity precipitously decreases with increasing salt concentration in a phenomenon termed as the polyelectrolyte effect. In this study, we provide evidence that the binding of early growth response (EGR) 1 transcription factor to DNA displays virtually zero dependence on ionic strength under physiological salt concentrations and that such feat is accomplished via favorable enthalpic contributions. Importantly, we unearth the molecular origin of such favorable enthalpy and attribute it to the ability of H382 residue to stabilize the EGR1-DNA interaction via both intermolecular hydrogen bonding and van der Waals contacts against the backdrop of salt. Consistent with this notion, the substitution of H382 residue with other amino acids faithfully restores salt-dependent binding of EGR1 to DNA in a canonical fashion. Remarkably, H382 is highly conserved across other members of the EGR family, implying that changes in bulk salt concentration are unlikely to play a significant role in modulating protein-DNA interactions central to this family of transcription factors. Taken together, our study reports the first example of a eukaryotic protein-DNA interaction capable of overriding the polyelectrolyte effect.

Role of promoter DNA sequence variations on the binding of EGR1 transcription factor.

In response to a wide variety of stimuli such as growth factors and hormones, EGR1 transcription factor is rapidly induced and immediately exerts downstream effects central to the maintenance of cellular homeostasis. Herein, our biophysical analysis reveals that DNA sequence variations within the target gene promoters tightly modulate the energetics of binding of EGR1 and that nucleotide substitutions at certain positions are much more detrimental to EGR1-DNA interaction than others. Importantly, the reduction in binding affinity poorly correlates with the loss of enthalpy and gain of entropy-a trend indicative of a complex interplay between underlying thermodynamic factors due to the differential role of water solvent upon nucleotide substitution. We also provide a rationale for the physical basis of the effect of nucleotide substitutions on the EGR1-DNA interaction at atomic level. Taken together, our study bears important implications on understanding the molecular determinants of a key protein-DNA interaction at the cross-roads of human health and disease.

Molecular determinants of the binding specificity of BH3 ligands to BclXL apoptotic repressor.

B-cell lymphoma extra-large protein (BclXL) serves as an apoptotic repressor by virtue of its ability to recognize and bind to BH3 domains found within a diverse array of proapoptotic regulators. Herein, we investigate the molecular basis of the specificity of the binding of proapoptotic BH3 ligands to BclXL. Our data reveal that while the BH3 ligands harboring the LXXX[A/S]D and [R/Q]XLXXXGD motif bind to BclXL with high affinity in the submicromolar range, those with the LXXXGD motif afford weak interactions. This suggests that the presence of a glycine at the fourth position (G+4)--relative to the N-terminal leucine (L0) within the LXXXGD motif--mitigates binding, unless the LXXXGD motif also contains arginine/glutamine at the -2 position. Of particular note is the observation that the residues at the +4 and -2 positions within the LXXX[A/S]D and [R/Q]XLXXXGD motifs appear to be energetically coupled-replacement of either [A/S]+4 or [R/Q]-2 with other residues has little bearing on the binding affinity of BH3 ligands harboring one of these motifs. Collectively, our study lends new molecular insights into understanding the binding specificity of BH3 ligands to BclXL with important consequences on the design of novel anticancer drugs.

Forward problem of electrocardiography based on cardiac source vector orientations.

To localize the unusual cardiac activities non-invasively, one has to build a prior forward model that relates the heart, torso, and detectors. This model has to be constructed to mathematically relate the geometrical and functional activities of the heart. Several methods are available to model the prior sources in the forward problem, which results in the lead field matrix generation. In the conventional technique, the lead field assumed the fixed prior sources, and the source vector orientations were presumed to be parallel to the detector plane with the unit strength in all directions. However, the anomalies cannot always be expected to occur in the same location and orientation, leading to misinterpretation and misdiagnosis. To overcome this, the work proposes a new forward model constructed using the VCG signals of the same subject. Furthermore, three transformation methods were used to extract VCG in constructing the time-varying lead fields to steer to the orientation of the source rather than just reconstructing its activities in the inverse problem. In addition, the unit VCG loop of the acute ischemia patient was extracted to observe the changes compared to the normal subject. The abnormality condition was achieved by delaying the depolarization time by 15ms. The results involving the unit vectors of VCG demonstrated the anisotropic nature of cardiac source orientations, providing information about the heart's electrical activity.

Cardiac computed tomography post-transcatheter aortic valve replacement.

Transcatheter aortic valve replacement (TAVR) is performed to treat aortic stenosis and is increasingly being utilised in the low-to-intermediate-risk population. Currently, attention has shifted towards long-term outcomes, complications and lifelong maintenance of the bioprosthesis. Some patients with TAVR in-situ may develop significant coronary artery disease over time requiring invasive coronary angiography, which may be problematic with the TAVR bioprosthesis in close proximity to the coronary ostia. In addition, younger patients may require a second transcatheter heart valve (THV) to 'replace' their in-situ THV because of gradual structural valve degeneration. Implantation of a second THV carries a risk of coronary obstruction, thereby requiring comprehensive pre-procedural planning. Unlike in the pre-TAVR period, cardiac CT angiography in the post-TAVR period is not well established. However, post-TAVR cardiac CT is being increasingly utilised to evaluate mechanisms for structural valve degeneration and complications, including leaflet thrombosis. Post-TAVR CT is also expected to have a significant role in risk-stratifying and planning future invasive procedures including coronary angiography and valve-in-valve interventions. Overall, there is emerging evidence for post-TAVR CT to be eventually incorporated into long-term TAVR monitoring and lifelong planning.

Molecular origin of the binding of WWOX tumor suppressor to ErbB4 receptor tyrosine kinase.

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in downregulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, nonconsensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of the WW1 domain of WWOX. Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX in agreement with our findings reported previously. Altogether, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes.

Biophysical basis of the promiscuous binding of B-cell lymphoma protein 2 apoptotic repressor to BH3 ligands.

B-cell lymphoma protein 2 (Bcl2) apoptotic repressor carries out its function by virtue of its ability to bind to BH3 domains of various pro-apoptotic regulators in a highly promiscuous manner. Herein, we investigate the biophysical basis of such promiscuity of Bcl2 toward its cognate BH3 ligands. Our data show that although the BH3 ligands harboring the LXXXAD motif bind to Bcl2 with submicromolar affinity, those with the LXXX[G/S]D motif afford weak interactions. This implies that the replacement of alanine at the fourth position (A + 4)-relative to the N-terminal leucine (L0) within the LXXXAD motif-to glycine/serine results in the loss of free energy of binding. Consistent with this notion, the A + 4 residue within the BH3 ligands harboring the LXXXAD motif engages in key intermolecular van der Waals contacts with A149 lining the ligand binding groove within Bcl2, whereas A + 4G/S substitution results in the disruption of such favorable binding interactions. Of particular interest is the observation that although increasing ionic strength has little or negligible effect on the binding of high-affinity BH3 ligands harboring the LXXXAD motif, the binding of those with the LXXX[G/S]D motif in general experiences a varying degree of enhancement. This salient observation is indicative of the fact that hydrophobic forces not only play a dominant but also a universal role in driving the Bcl2-BH3 interactions. Taken together, our study sheds light on the molecular basis of the factors governing the promiscuous binding of Bcl2 to pro-apoptotic regulators and thus bears important consequences on the development of rational therapeutic approaches.

Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery.

Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner.

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues.