Capturing RAS oligomerization on a membrane.

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

Water Plays Key Roles in Stabilities of Wild Type and Mutant Transthyretin Complexes.

Transthyretin (TTR), a 56 kDa homotetramer that is involved in the transport of thyroxine and retinol, has been linked to amyloidosis through disassembly of tetramers to form monomers, dimers, and trimers that then reassemble into higher order oligomers and/or fibrils. Hybrid TTR (hTTR) tetramers are found in heterozygous individuals that express both wild type TTR (wt-TTR) and mutant TTR (mTTR) forms of the protein, and these states display increased rates of amyloidosis. Here we monitor subunit exchange (SUE) reactions involving homomeric and mixed tetramers using high resolution native mass spectrometry (nMS). Our results show evidence that differences in TTR primary structure alter tetramer stabilities, and hTTR products can form spontaneously by SUE reactions. In addition, we find that solution temperature has strong effects on TTR tetramer stabilities and formation of SUE products. Lower temperatures promote formation of hTTR tetramers containing L55P and V30M subunits, whereas small effects on the formation of hTTR tetramers containing F87A and T119M subunits are observed. We hypothesize that the observed temperature dependent stabilities and subsequent SUE behavior are a result of perturbations to the network of "two kinds of water": hydrating and structure stabilizing water molecules (Spyrakis et al. J. Med. Chem. 2017, 60 (16), 6781-6827; Xu et al. Soft Matter 2012, 8, 324-336) that stabilize wt-TTR and mTTR tetramers. The results presented in this work illustrate the utility of high resolution nMS for studies of the structures, stabilities, and dynamics of protein complexes that directly influence SUE reactions.

Native mass spectrometry and structural studies reveal modulation of MsbA-nucleotide interactions by lipids.

The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.

Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

2'-Deoxy Guanosine Nucleotides Alter the Biochemical Properties of Ras.

Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-RasG12C was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOScat) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOScat differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.

Changes in Eyebrow Position and Movement with Aging.

BACKGROUND: This study evaluated dynamic changes in eyebrow position related to aging. METHODS: Female participants were recruited and separated into two groups aged 20-30 years (the younger group, n=20; mean age, 24.8 years) and 50-70 years (the older group, n=20; mean age, 55.8 years). Photogrammetry was used to determine the eyebrow position at the medial canthus (MC), lateral limbus, lateral canthus, and lateral end point (EP) for 6 actions: smooth opening (the reference action) and closing of the eye, forward gaze, maximum opening and closing of the eye, and maximum frown. Videos were also recorded. RESULTS: No differences in eyebrow position were detected at the MC when opening or closing the eyes smoothly, gazing straight ahead, or closing the eyes maximally. For all 6 actions, the position of the lateral EP in the older group was significantly lower than in the younger group (P=0.003), and the smallest degree of vertical movement at this point was found in both age groups (P<0.001). Vertical movement at the 4 landmarks of the eyebrows decreased with aging. CONCLUSIONS: Eyebrow position was unchanged at the MC with aging, except at maximal eye opening and maximal frown. No differences in eyebrow position were observed between the younger and older groups when eyes were maximally closed, except at the EP. It is important to focus on correction of the lateral EP for periorbital rejuvenation.

Changes of eyebrow muscle activity with aging: functional analysis revealed by electromyography.

BACKGROUND: This study evaluated the activity of the frontalis muscle, the corrugator supercilii muscle, and the orbicularis oculi muscle according to eyebrow movement and aging. METHODS: Two random cohorts of women aged 20 to 30 years (young group, n = 20; mean age, 24.8 years) and 50 to 70 years (old group, n = 20; mean age, 55.8 years) were recruited prospectively. Surface electromyography was used to evaluate motor unit action potential for each muscle in each of six eyebrow movements. Details on statistical analysis are described in the text. RESULTS: In both age groups, corrugator supercilii muscle activity was the highest for all six movements. Frontalis muscle activity was highest with maximal frowning but not with maximal eye opening. Orbicularis oculi muscle activity was significantly greater in the older age group than in the younger age group for most actions. When the motion proportion of each muscle was compared, the respective orbicularis oculi muscle and corrugator supercilii muscle indices were higher in the older age group than in the young group. CONCLUSIONS: Eyebrows are maintained by the dynamic balance of frontalis, corrugator supercilii, and orbicularis oculi muscles, and various combinations of motor recruitment of these muscles determine the eyebrow position and shape. For youthful eyebrows, attenuation of the depressor muscle may restore the muscle balance in treatments for eyebrow rejuvenation.

Large auricular chondrocutaneous composite graft for nasal alar and columellar reconstruction.

BACKGROUND: Among the various methods for correcting nasal deformity, the composite graft is suitable for the inner and outer reconstruction of the nose in a single stage. In this article, we present our technique for reconstructing the ala and columella using the auricular chondrocutaneous composite graft. METHODS: From 2004 to 2011, 15 cases of alar and 2 cases of columellar reconstruction employing the chondrocutaneous composite graft were studied, all followed up for 3 to 24 months (average, 13.5 months). All of the patients were reviewed retrospectively for the demographics, graft size, selection of the donor site and outcomes including morbidity and complications. RESULTS: The reasons for the deformity were burn scar (n=7), traumatic scar (n=4), smallpox scar (n=4), basal cell carcinoma defect (n=1), and scar contracture (n=1) from implant induced infection. In 5 cases of nostril stricture and 6 cases of alar defect and notching, composite grafts from the helix were used (8.9×12.5 mm). In 4 cases of retracted ala, grafts from the posterior surface of the concha were matched (5×15 mm). For the reconstruction of the columella, we harvested the graft from the posterior scapha (9×13.5 mm). Except one case with partial necrosis and delayed healing due to smoking, the grafts were successful in all of the cases and there was no deformity of the donor site. CONCLUSIONS: An alar and columellar defect can be reconstructed successfully with a relatively large composite graft without donor site morbidity. The selection of the donor site should be individualized according to the 3-dimensional configuration of the defect.