Identification of degradation pathways for HSP90 client proteins

Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that plays critical roles in regulating the folding, stabilization, post-translational modification, activation and maturation of its various client proteins, of which many are oncoproteins. Impairing the function of HSP90 by the inhibition of its ATPase cycle with inhibitors such as AUY922 promotes the ubiquitylation and proteasomal degradation of its client proteins. However, we currently do not fully understand the mechanism for ATPase-inhibited triggered degradation of client proteins, and which E3 ligase systems are involved. Although previous studies revealed a number of E3 ligases including CHIP and CUL5 as potentially E3 ligases involved in the degradation of HSP90-dependent client protein, these have often used cancer cells that may have dysregulated systems. Additionally, other components of such E3 ligase systems have not been well characterised. Using a Reverse Transfection Format (RTF) siRNA screen system we identified two E3 ligases that are involved in two independent pathways for mediating proteasomal degradation of the HSP90-dependent protein kinase CRAF in HEK293 cells. The elongin BC-CUL5-SOCS-box protein (ECS) complex operates one pathway for the degradation of CRAF, while a novel but poorly described HECTD3 from the HECT-family was identified as the main E3 ligase for degrading CRAF following the pharmaceutical inhibition of HSP90. We revealed a potential complexes consisting of CRAF, HSP90 and HECTD3, which may contribute towards identifying the pathway for the degrading of such HSP90-dependent client protein kinases. We were also able to show that depriving access of CRAF to CDC37 and therefore HSP90 resulted in an HECTD3 and CUL5 independent degradation pathway. These studies form the basis of establishing the complex network of pathways that help to regulate CRAF protein levels

2-(2-Nitro­phen­yl)-1,3-dioxan-5-ol

In the title compound, C10H11NO5, the six-membered 1,3-dioxane ring displays a chair conformation, with the hydr­oxy and 2-nitro­phenyl groups in equatorial positions, which minimizes steric hindrance. In the crystal, mol­ecules are linked into chains along the b axis by inter­molecular O—H⋯O hydrogen bonds

(S)-3-Acetyl-3-[(R)-1-(4-bromo­phen­yl)-2-nitro­eth­yl]oxolan-2-one

The title compound, C14H14BrNO5, has two chiral C atoms. The quaternary C atom in the oxolanone ring has an S configuration, while the adjacent tertiary C atom has an R configuration. The oxolanone ring adopts an envelope conformation, with the flap C atom lying 0.298 (3) Å from the mean plane of the remaining four atoms. In the crystal, mol­ecules are connected into chains along [010] via weak C—H⋯O hydrogen bonds

HECTD3 mediates an HSP90-dependent degradation pathway for protein kinase clients

Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway

(S)-2-[(S,E)-4-(4-Chloro­phen­yl)-1-nitro­but-3-en-2-yl]cyclo­hexa­none

The title compound, C16H18ClNO3, was obtained by the organocatalytic asymmetric Michael addition of cyclo­hexa­none to 1-chloro-4-[(1E,3E)-4-nitro­buta-1,3-dien­yl]benzene. The double bond has an E configuration. The cyclo­hexa­none ring adopts a chair conformation. The conformation of the mol­ecule is stabilized by a weak intra­molecular C—H⋯O hydrogen bond

Ethyl (2R,3S)-2-benzoyl-3-(4-bromo­phen­yl)-4-nitro­butano­ate

The title compoud, C19H18BrNO5, was synthesized by an organocatalytic reaction. The aymmetric unit contains two independent mol­ecules, in each of which the carbon between the two carbonyl groups adopts an R configuration, while the adjacent C atom has an S configuration. The dihedral angle between the two benzene rings is different in the two mol­ecules [11.64 (3) and 58.96 (4)°]

Subsequent monitoring of ferric ion and ascorbic acid using graphdiyne quantum dots-based optical sensors

Graphdiyne (GDY) as an emerging carbon nanomaterial has attracted increasing attention because of its uniformly distributed pores, highly π-conjugated, and tunable electronic properties. These excellent characteristics have been widely explored in the fields of energy storage and catalysts, yet there is no report on the development of sensors based on the outstanding optical property of GDY. In this paper, a new sensing mechanism is reported built upon the synergistic effect between inner filter effect and photoinduced electron transfer. We constructed a novel nanosensor based upon the newly-synthesized nanomaterial and demonstrated a sensitive and selective detection for both Fe3+ ion and ascorbic acid, enabling the measurements in real clinical samples. For the first time fluorescent graphdiyne oxide quantum dots (GDYO-QDs) were prepared using a facile ultrasonic protocol and they were characterized with a range of techniques, showing a strong blue-green emission with 14.6% quantum yield. The emission is quenched efficiently by Fe3+ and recovered by ascorbic acid (AA). We have fabricated an off/on fluorescent nanosensors based on this unique property. The nanosensors are able to detect Fe3+ as low as 95 nmol L−1 with a promising dynamic range from 0.25 to 200 μmol L−1. The LOD of AA was 2.5 μmol L−1, with range of 10–500 μmol L−1. It showed a promising capability to detect Fe3+ and AA in serum samples