El centro psiquiátrico en los Estados Unidos del siglo XX

Included in the functions of HDACs is the regulation of many other proteins either through direct acetylation or through scaffolding interactions. HDACs in-turn are regulated through a number of different processes at many levels, from transcrip-tional repression to a variety of PTMs. Elucidation of the function and mechanism of action of these regulating modifications could help in understanding which parts of the protein are flexible and amenable to activity altering structural changes. With this knowledge more effective rational drug design can be undertaken by targeting naturally occuring regulatory systems. Elevated HDAC8 expression is correlated

with advanced disease progression and is a negative prognostic indicator for patients with neuroblastoma⁷⁶. Direct transcriptional control of HDAC8 has been demon-strated by SOX4, a direct target gene of FRA-2 which causes elevated expression of HDAC8 in adult T-cell leukemia/lymphoma⁹⁸.

1.3.1 Regulation of subcellular location

Substrate availability, and subsequent activity of an enzyme, can vary greatly de-pending on cellular compartment. Unlike HDAC1, HDAC2 and HDAC3, which are exclusively nuclear proteins, HDAC8 is often detected outside the nucleus^24,53. It follows therefore that HDAC8 has non-nuclear substrates and/or interaction part-ners. 14-3-3 proteins are chaperones which bind some phosphorylated proteins (such as transcription factors) and translocate them into the nucleus; HDAC4, HDAC5 and HDAC7 have been identified as targets that are shuttled in this way⁹⁹. HDAC8 has a nuclear localisation sequence⁷ and may also shuttle between the nucleus and cytoplasm in a similar fashion as it has been observed as both phosphorylated and non-phosphorylated forms within cells¹⁰⁰.

Unlike all other HDACs (with the exception of HDAC11), HDAC8 has no regu-latory sequences or domains outside its core catalytic domain. The lack of these additional sequences and domains in HDAC8 suggests a more promiscuous func-tionality as these additional domains and sequences are a major part of selectivity in HDACs by enabling their recruitment to complexes and stabilise and strengthen protein-protein interactions. Redundancies and compensatory mechanisms in many signalling pathways also make substrate detection difficult as observed effects may not be as a direct result of HDAC8 action but a knock-on effect. Currently it is unknown what mechanisms regulate the subcellular location of HDAC8, under-standing of these mechanisms may provide new directions for HDAC8 inhibition, perhaps even through indirect inhibition or activation of the HDAC8-interacting protein which regulates subcellular location.

1.3.2 Post translational modifications

Phosphorylation of HDAC1 and HDAC2 by casein kinase II (CK2) is an activating PTM which promotes incorporation into active complexes such as mSin3A and CoR-EST^8,41, conversely, phosphorylation of HDAC8 has a downregulating effect. Despite having 19 predicted possible phosphorylation sites, HDAC8 is only observed to be phosphorylated at one site, Ser39, both in vitro and in vivo. Ser39 phosphorylation is only performed by cyclic adenosine monophosphate (cAMP) dependent protein kinase A (PKA) and HDAC8 is the only HDAC with a PKA phosphorylation mo-tif¹⁰⁰. Ser39 is conserved in HDAC8 but not found in other HDACs and so this inactivation by PKA is unique to HDAC8.

Ser39 is located 21˚A from the catalytic centre of HDAC8, the inhibition there-fore, cannot be a result of a simple blocking of catalysis or substrate binding. As with many other PTMs, the regulation is most likely achieved through an allosteric effect. Ser39 lies in a hydrophobic pocket so its phosphorylation may cause a struc-tural rearrangement of the protein due to the additional charge imparted by the phosphate⁸². A possible explanation for the regulation involves Arg37, the acetate release “gatekeeper”¹⁰¹. Ser39 sits on the same α-helix as Arg37 (see Figure 1.8) and phosphorylation may “shut the gate”, thereby inhibiting subsequent deacetylation reactions. Molecular dynamics simulations of HDAC8 suggest that Arg37 forms a barrier with backbone carbonyl groups restricting the access of water to the active site and the escape of acetate¹⁰¹.

Fifteen HDAC8 interacting proteins have been identified using a bacterial two-hybrid experiment; of which, six required HDAC8 phosphorylation⁵². This strongly implies that phosphorylation is key to some HDAC8 protein-protein interactions whether the phosphorylation is of HDAC8 itself or of the interacting protein. If the structural basis for this inhibition was well understood it may aid in targeting of an inhibitor that binds a stable, rigid conformation of HDAC8, possibly in a non competitive manner.

Acetylation and glycosylation are also possible HDAC8 regulatory mechanisms. The NetNGlyc 1.0 server identifies two potential glycosylation sites but does not predict

Arg37

Ser39

K⁺ Zn²⁺

4

Figure 1.8: HDAC8-4 structure, two bound K⁺, the catalytic Zn²⁺, Ser39 and Arg37, which are all important in HDAC8 regulation are shown.

glycosylation of either site¹⁰². HDAC1 has two acetylation sites in the catalytic domain which inhibit activity towards histones¹⁰³. One of these sites is conserved in HDAC8 and is close to the inhibitory monovalent cation binding site¹⁰⁴ (see Section 1.3.4), this has been suggested to be one possible mode of HDAC8 regulation but has yet to be tested²⁶. PTMs within HDACs can play an important part in drug binding, a PTM can have a dramatic effect on the protein structure and could affect a drug binding region by completely abolishing binding or conversely cause structural change that facilitates drug binding. For example if an inhibitor could be designed to stabilise the conformation of phospho-HDAC8 through binding in this region it would be a non-competitive and highly selective HDACi.

1.3.3 Activation by small molecules

All known HDACi are competitive inhibitors which block access to the active site.

Recently N -acetylthioureas have been shown to function as activators of HDAC8¹⁰⁵. This was determined to be through a 5-fold increase in kcat and lowering of the KM

by increasing the power of aromatic stacking interactions which help to bind the substrate to the mouth of substrate tunnel (determined by docking simulations).

In a physiological context some small molecule metabolites have also been found to stimulate HDAC1 and HDAC2 activity in a very specific manner such as coenzyme A derivatives and NADPH (but not NADP⁺, NADH or NAD⁺)¹⁰⁶. Inositol phosphates have been shown to have a role in the regulation of HDAC1, HDAC2 and HDAC3, in particular, recent structures reveal the structural basis for this regulation whereby inositol phosphates mediate the formation of the HDAC complexes HDAC1-MTA1¹⁰⁷ and HDAC3-NcoR2⁸⁷. Inositol phosphates and other metabolites do not have an effect on HDAC8 but these types of protein-protein contact mediating molecule sites may prove useful if they are discovered on HDAC8 in future by identyfying sites that can be targeted in the design of inhibitors. These sorts of molecules may be useful in diseases correlated with low HDAC activity (eg. HDAC1 and HDAC3 in metastatic melanoma¹⁰⁸) where an increase in HDAC activity may be beneficial to a patient.

1.3.4 Regulation by cations

Divalent and monovalent metal ions have been shown to have an effect on HDAC8 activity, they modulate the kinetics of catalysis but also possibly regulate activity through other mechanisms which are not fully understood.

The catalytic Zn²⁺ in recombinantly expressed HDAC8 has been successfully ex-changed with Co²⁺, Fe²⁺, Ni²⁺ and Mn²⁺¹⁰⁹. Fe²⁺ containing HDAC8 has catalytic efficiency 2.8-fold greater than HDAC8-Zn²⁺. It is possible that HDAC8 may exist naturally as a mixture of Fe²⁺ and Zn²⁺ bound enzyme as the approximate KD val-ues of both ions for HDAC8 (5-400 pM vs 0.2-6 µM respectively) are in the region of the estimated cellular concentrations (5-400pM vs 0.2-0.6 µM respectively)¹¹⁰. Due to difficulties expressing large amounts of other HDACs detailed analysis of the potential active site metal ions has not been undertaken. Cellular concentrations of Zn²⁺ can change significantly in response to oxidative^111,112 and metal toxicity stresses¹¹³, HDAC8 activity may be regulated in part through these ionic fluxes.

In all HDAC8 crystal structures, two monovalent cations (MVC) are bound (see Figure 1.8) which have been demonstrated to have different functions. Binding at one site is activating¹¹⁴ while the other is an inhibiting¹⁰⁴. With peak activity occuring around the cellular concentrations of K⁺(typically 139 mM¹¹⁵) it is possible that binding of fluctuations in K⁺ concentration is a in vivo inhibitor of HDAC8.

These sites are conserved across class I and class II HDACs so this mechanism of regulation may not be unique to HDAC8¹⁰⁴

HDAC8 activity is likely regulated by a combination of processes: concentrations of intracellular Zn²⁺, Fe²⁺, K⁺ and Na⁺, phosphorylation of Ser39 by PKA (which is itself activated by a number of different pathways) and interactions with proteins to localise HDAC8 with substrates, binding partners or to a cellular compartment.