Photocontrol of GTPase Cycle and Multimerization of the Small G-Protein H-Ras using Photochromic Azobenzene Derivatives

Ras is a small G protein known as a central regulator of cellular signal transduction that induces processes, such as cell division, transcription. The hypervariable region (HVR) is one of the functional parts of this G protein, which induces multimerization and interaction between Ras and the plasma membrane. We introduced two highly different in polarity photochromic SH group-reactive azobenzene derivatives, N-4-phenyl-azophenyl maleimide (PAM) and 4-chloroacetoamido-4-sulfo-azobenzene (CASAB), into three cysteine residues in HVR to control Ras GTPase using light. PAM stoichiometrically reacted with the SH group of cysteine residues and induced multimerization. The mutants modified with PAM exhibited reversible changes in GTPase activity accelerated by the guanine nucleotide exchange factor and GTPase activating protein and multimerization accompanied by cis- and trans-photoisomerization upon ultraviolet and visible light irradiation. CASAB was incorporated into two of the three cysteine residues in HVR but did not induce multimerization. The H-Ras GTPase modified with CASAB was photo controlled more effectively than PAM-H-Ras. In this study, we revealed that the incorporation of azobenzene derivatives into the functional site of HVR enables photo reversible control of Ras function. Our findings may contribute to the development of a method to control functional biomolecules with physiologically important roles.

Noncanonical structural requirements of neurofibromin SUMOylation reveal a folding-deficiency of several pathogenic mutants

Neurofibromin (Nf1) is a large multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a frequently occurring genetic disease, neurofibromatosis type I, and in various cancers. The best described function of Nf1 is its Ras-GTPase activity, carried out by its GAP-related domain (GRD). SecPH, another structurally well-characterized domain of Nf1, is immediately adjacent to the GRD and interacts with lipids and proteins, thus connecting Nf1 to diverse signalling pathways. Here, we demonstrate, for the first time, that Nf1 and SecPH are substrates of the SUMO pathway. We identified a well-defined SUMOylation profile of SecPH and a main SUMOylation event on Lys1731 that appears to play a role in Ras-GAP activity. Our data allowed us to characterize a new set of pathogenic Nf1 missense mutants that exhibits a disrupted SUMOylation profile that may correlate with their unfolding. Accordingly, Lys1731 SUMOylation is mediated by a noncanonical structural motif, therefore allowing a read-out of SecPH conformation and folding status.

Ras Isoforms from Lab Benches to Lives—What Are We Missing and How Far Are We?

The central protein in the oncogenic circuitry is the Ras GTPase that has been under intense scrutiny for the last four decades. From its discovery as a viral oncogene and its non–oncogenic contribution to crucial cellular functioning, an elaborate genetic, structural, and functional map of Ras is being created for its therapeutic targeting. Despite decades of research, there still exist lacunae in our understanding of Ras. The complexity of the Ras functioning is further exemplified by the fact that the three canonical Ras genes encode for four protein isoforms (H-Ras, K-Ras4A, K-Ras4B, and N-Ras). Contrary to the initial assessment that the H-, K-, and N-Ras isoforms are functionally similar, emerging data are uncovering crucial differences between them. These Ras isoforms exhibit not only cell–type and context-dependent functions but also activator and effector specificities on activation by the same receptor. Preferential localization of H-, K-, and N-Ras in different microdomains of the plasma membrane and cellular organelles like Golgi, endoplasmic reticulum, mitochondria, and endosome adds a new dimension to isoform-specific signaling and diverse functions. Herein, we review isoform-specific properties of Ras GTPase and highlight the importance of considering these towards generating effective isoform-specific therapies in the future.

Mobility of the gradient tracking machine in mating yeast depends on Bud1 inactivation and actin-independent vesicle delivery

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through the secretion of mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Because strong positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking after its assembly is complete. What prevents the GTM from triggering polarized growth at the DS? Here we describe two mechanisms that enable tracking. First, the Ras GTPase Bud1 must be inactivated to release the GTM. Second, actin-independent – but not actin-dependent – vesicle delivery must be targeted upgradient to effect GTM redistribution.

Expression of human G3BP1 in E. coli

G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein) is responsible for normal RNA stress granule (SG) assembly and overexpressed in many cancer cells. Deletion of G3BP1 decreases the number and size of SGs. SGs are complex of RNA and proteins that stall translation of protein in response to stress. Given the function of G3BP1 in stress granule assembly and tumor suppression, it is believed that G3BP1 regulates cell growth and proliferation as well. Here, I constructed the recombinant protein expression vector and systemically optimized condition for the expression of human G3BP1 protein in E. coli. This research should be useful for investigating further functional analysis and atomic structure of G3BP1.