SCF substrates

The accumulation of SCF substrates in CSN mutants suggests that neddylation of Cull may be required for the repeated cycles of ubiquitin transfer in vivo. 

Despite the identification of SCF as the ligase that ubiquitinates p27 in vivo, ubiquitination of p27 in vitro using purified components of the specific ubiquitination machinery could not be reconstituted until recently. In contrast, ubiquitination of the yeast SCF substrate, Sid, as well as ubiquitination of iKfia by human can be achieved in vitro... 

Enzyme source or commercial name SCF Substrates Conditions Refer- ence... 

Adsorption and Desorption Adsorbents may be used to recover solutes from supercritical fluid extracts for example, activated carbon and polymeric sorbents may be used to recover caffeine from CO9. This approach may be used to improve the selectivity of a supercritical fluid extraction process. SCF extraction may be used to regenerate adsorbents such as activated carbon and to remove contaminants from soil. In many cases the chemisorption is sufficiently strong that regeneration with CO9 is limited, even if the pure solute is quite soluble in CO9. In some cases a cosolvent can be added to the SCF to displace the sorbate from the sorbent. Another approach is to use water at elevated or even supercritical temperatures to facilitate desorption. Many of the principles for desorption are also relevant to extraction of substances from other substrates such as natural products and polymers. 

The SCF ubiquitylates proteins from late Gl to early M phase. This complex consists of a core together with different so called F-box proteins, which are responsible for the substrate recognition. Typically... 

The solubility of a solute in scC02 is extremely dependent on its structure, with three features of paramount importance. As expected, compounds of low polarity are more soluble than very polar compounds or salts. However, solubility also increases greatly with increasing vapour pressure of a substrate. To account for the contribution of volatility and solvation to the solubility process, Kurt Zosel coined the term Destraktion (from Latin destillare and extrahere) in his pioneering work on natural product extraction with SCFs [5], Finally, some specific functional groups like perfluoroalkyl and polysiloxane substituents, or polyether/polycarbonate copolymers... 

As with classical multiphase catalysis, the organometallic catalyst is retained here in a liquid phase that is immiscible with the second phase containing substrates and/or products. For hydrogenation, the liquid/SCF system is always biphasic, whereas conventional systems are usually triphasic (liquid-1 /liquid-2/ H2). The liquid phase must provide a stable environment for the organometallic catalyst and should be insoluble in the SCF phase. Water, ILs and PEG have been used successfully for this purpose, together with scC02 as the mobile phase. Again, the products must not be too polar in order to be effectively extracted if C02 is used as the SCF. 

Biphasic systems that contain the catalyst in the supercritical phase and the substrates/products in a second liquid phase can also be implemented. With water as the polar phase, these inverted systems are particularly attractive for the conversion of highly polar and/or low-volatile hydrophilic substrates with limited solubility in typical SCFs such as scC02. 

Skowyha, D., et al., F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell, 1997, 91(2), 209-19. 

Deeeenbaugh, a. E., et al.. Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sicl. Cell, 2003,... 

A model of the full SCF/E2 complex [112] shows that the end of Skp2 which binds the substrate is pointed toward the Rbxl-bound E2, with a 50-A gap between the two. Models based on two other SCF structures show similar distances between the F-box protein and the E2 [109, 115]. Whether this gap can be bridged by the bound substrate is currently unclear. It has been suggested that the E2 may bind to Rbxl somewhat differently than UbcH7 is observed to bind in the c-Cbl RING/ UbcH7 complex, but it is not obvious that this can lead to a 20 A movement of the E2 toward the bound substrate as suggested [109]. 

The essential components of the SCF ubiquitin E3 ligase include Skpl, Cul-1/ Cdc53, one of many F-box proteins, and the RINC-H2-finger protein Rod (Rbxl or Hrtl) (Figure 6.2). Although initial studies did not reveal the presence of a fourth component of the SCF complex [14, 15], later work showed that a RINC-H2-finger protein, Rod, is an essential subunit of the SCF complex [3]. The SCF complex thus contains three invariable components (Rod, Cull, and Skpl) which provide a core structure to which one of the many substrate-specific subunits (F-box proteins) binds. The Rocl-Cull-Skpl core also independently interacts with the ubiquitin E2-conjugating enzyme to couple ubiquitin transfer to the substrates [3]. One of the E-box proteins binds directly to a specific substrate and such interaction facilitates the polyubiquitination of the substrate by ubiquitin... 

Fig. 6.2. The structures of SCF, Cul2-Elongin B-C, and Cul3-BTB/POZ ubiquitin E3 ligase complexes with the bound substrates and E2 enzymes.

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