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Lesch-Nyhan Disease: A Moonlighting Example?

March 18, 2018

Previous posts of Biopolyverse (particularly that of August 2016) have considered the phenomenon of protein ‘moonlighting’, where many proteins have been well documented as possessing more than one distinct functional role. In this post, an example is considered where moonlighting is a possible explanation of a striking phenotype associated with a specific genetic lesion. The gene in question is HPRT1, encoding the enzyme hypoxanthine-guanine-phosphoribosyl transferase, which is important in cellular purine salvage pathways.

Terrible but Fascinating

In 1964, William Nyhan and Michael Lesch published a description of a hitherto unrecognized genetic disease, which has eponymously become known as Lesch-Nyhan syndrome, or Lesch-Nyhan disease (LND). It was later shown that this condition was characterized by the genetic loss of a specific enzyme, called hypoxanthine-guanine-phosphoribosyl transferase. (This has been abbreviated in various ways, including HGPRT, HGPRTase, and HPRT. For simplicity, the later term will be used here to refer to this enzyme). The gene encoding HPRT (HPRT1) is situated on the X-chromosome, and as a sex-linked gene, its functional loss is almost exclusively seen in males.

What does HPRT, in its role as a protein catalyst, normally do? It is actually well understood as a key mediator within a metabolic pathway which allows purines (chemical components of A and G nucleotides that are, along with pyrimidines, fundamental in DNA and RNA structure) to be recycled back into active biochemical function. This process is thus called a ‘salvage’ pathway, and is seen ubiquitously across all domains of life.  In humans a defective purine salvage pathway results in the accumulation of uric acid, which can cause gout and liver failure. Although this is indeed a feature of Lesch-Nyhan disease, it is not the known biochemical enzymology of HPRT which creates the compelling interest in this condition.  Since HPRT is expressed in all nucleated cells, it might be expected from first principles that its profound deficiency would not have particular organ-specific effects. And yet it most certainly does, in the nervous system, where a range of cognitive impairments are seen with afflicted children. Even this is not as striking as the ‘behavioral phenotype’ of self-mutilation, which is a hallmark of ‘classic’ LND.  This self-injurious behavior (SIB) can take the form of severe lip and finger biting, or other forms of self-damage, which have been graphically described in the literature. As a relentlessly compulsive affliction, it is a terrible burden on both victims and their carers, without any truly effective treatment. Thus, as many have noted before, loss of a single gene product can evidently have a profound effect on human behavior. While self-harming behaviors arising from genetic mutations in humans are not unique to LND, it is especially pronounced in the latter, and one diagnostic criterion for the ‘classic’ form of the disease. There are many ramifications of these observations for neurology, psychology, and even philosophy, all of which combine to produce an extra element of compelling interest into this compulsive disorder.

Abbreviations

De novo vs. Salvage

In general terms, a salvage enzyme can be seen as an energy-saving back-up, in order to promote the efficiency of house-keeping operations in any biosystem by recycling precursor compounds. As such, salvage has its own biological and evolutionary logic, and is accordingly a ubiquitous biological feature. The purine salvage processes mediated (in part) by HPRT are depicted in Fig. 1 below.

Fig1F-SalvagePaths

Fig. 1. HPRT and purine salvage pathways. Both the purines hypoxanthine and guanine are substrates for HPRT catalysis, with the additional requirement for 5’-phosphoribosyl pyrophosphate (PRPP) to provide an activated ribose moiety towards the formation of purine nucleotides. Guanosine monophosphate (GMP) is made directly from guanine and PRPP via the catalytic auspices of HPRT, while hypoxanthine as substrate requires additional enzymatic processing to yield either GMP or adenosine monophosphate (AMP). AMP is also produced by the activity of a different salvage enzyme, adenine phosphoribosyl transferase (APRT), which acts on adenine plus PRPP.

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Human HPRT is a protein of 217 amino acid residues (Fig. 2), which requires magnesium for its catalytic activity, and is normally found as a tetramer (4 subunits of the monomeric protein).

Fig.2-HumHPRTFig. 2.  Structure of a monomer of human HPRT in complex with guanosine monophosphate (GMP). This image has been generated from Protein Databank entry 1HMP. Here alpha-helices, beta-sheets, and turns are indicated by red, green, and blue segments respectively.

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A salvage pathway can be distinguished from synthesizing a biochemical compound from scratch, usually termed ‘de novo’ synthesis (from Latin, ‘of new’). Most organisms possess both synthetic approaches towards purines in their biochemical repertoires, such is its fundamental biological importance. Humans are certainly included among species which can synthesize their own purines as well as recycle them via salvage pathways. The de novo synthesis of purines is well-defined, involving numerous enzymatic steps, which result in the formation of multienzyme complexes termed ‘purinosomes’ under conditions of high purine demand.

Another moonlighting enzyme?

Following this background, we have seen that the enzymatic activity of HPRT is well-understood at the molecular level. So why should it be a candidate for having a functional moonlighting role, in addition to its normal catalytic task? Before commenting further, it will be useful to note the general meaning of moonlighting in this context, with reference also back to a previous post on this topic. A second functional role for an enzyme if true moonlighting is involved implies that the enzyme performs some function quite distinct from its normal catalysis. Thus, HPRT is sometimes noted as having two roles in terms of its ability to utilize more than one substrate (both guanine and hypoxanthine), but this is not moonlighting in itself. A moonlighting second function would necessarily involve something unrelated to the activity towards either conventional HPRT substrate (Fig. 1), which could also be quite distinct from purine salvage entirely. In general terms, a moonlighting function for any enzyme will necessarily involve interactions with other biomolecules beyond its conventional substrates or cofactors. Since an enzyme’s catalytic center is usually evolutionarily fine-tuned for a specific activity, a second functional site will logically be more likely found on a distinct domain of the same protein. This kind of functional separation has been observed with known polyfunctional proteins, but overlap between functional domains may also occur.

At first glance, imputing a moonlighting role to HPRT might seem quite rational, given the striking neurological phenotype of Lesch-Nyhan victims and the apparent difficulty of accounting for how absence of salvage enzyme function alone could bring about such higher-level behavioral abnormalities. Indeed, the possibility of a moonlighting role for HPRT in itself it is not an original suggestion with this post, and it would be quite surprising if it was. Yet the consideration of hypothetical HPRT moonlighting does not seem to have been much pursued in the literature.

There is in fact a likely reason for this apparent neglect, and that stems from a plausible manner in which HPRT deficiency could have deep ramifications beyond merely ensuring that DNA and RNA synthesis is kept up to speed.

Guanosine nucleotides – a definite dual role

As a term in biology, moonlighting is generally applied to proteins, but it is possible to extend its ambit broadly in general biosystems, in line with the notion of biological parsimony (internal ref). One such example can be found which is strongly relevant here, and that concerns the biology of guanosine nucleotides.

A very large and diverse family of mammalian cell surface receptors are called GPCRs, for G-Protein Coupled Receptors. As the name implies, these receptors use guanosine nucleotides (GDP and GTP) as part of a molecular on-off switch for the signaling process. So, in a truly parsimonious manner, guanosine nucleotides play a fundamental role in a wide variety of essential signaling transduction pathways, as well as being basic structural and informational constituents of nucleic acids. The possible relevance of this to the Lesch-Nyhan phenotype comes from two pieces of information: (1) the neurotransmitter dopamine acts as a ligand for five separate receptors, all of the GPCR class, and (2) perturbations in dopamine-mediated (dopaminergic) neural pathways have been reported in LND and associated experimental cellular models. The linking of these observations with the LND phenotype comes from the reasonable proposition that deficiency in the purine salvage pathway leads to a corresponding shortfall in neural GDP / GTP pools, with consequent disarray in certain (principally dopaminergic) neural signaling pathways. In turn, by this proposal such abnormalities ultimately result in the over-riding of the normal avoidance of self-destructive behavior.

A problem with this interpretation comes from direct measurement of guanosine nucleotide pools in normal vs. LND neural tissues. If salvage deficiency meant that neurones cannot maintain sufficient levels of purine nucleotides, it should be quantifiable, and yet experimentation has not borne this out. Defects in the salvage pathway trigger up-regulation of de novo purine synthesis, evidenced by high purinosome levels in Lesch-Nyhan cells, irrespective of disease severity. As a counter-argument to this, it has been suggested that de novo synthesis may be nevertheless unable to cope with certain circumstances of unusually high purine demand placed on certain neural cells in particular, given their requirements for GTP / GDP in signaling as well as nucleic acid turnover. Yet they are not only cell types with this extra requirement.

As an alternative possibility, it has been proposed that excessive de novo purine production in the absence of salvage pathways may lead to the accumulation of neurotoxic by-products, but this has not been well-substantiated. In that general vein, it has long been known that treatment of LND patients with an inhibitor of uric acid formation alleviates gout-like symptoms, but without effect on the SIB phenotype.

Patterns of mutations

In considering the possibility of a moonlighting role for HPRT, it is necessary to look at the possible effects of mutations in a protein’s amino acid sequence that could arise as a consequence of human germline mutations at the DNA level. Obviously, large deletions or rearrangements could destroy the entire coding sequence for a protein, and all possible functional activities of it would be irrevocably lost. The effects of single amino acid residue substitutions could range from protein misfolding and aggregation or degradation associated with global low activity, to having little or no bearing on effective function. In between these two extremes are mutations which are compatible with global protein folding (albeit with possible reduced stability), while associated with localized functional loss. This is depicted schematically in Fig. 3 below.

Fig3-MoonlightingProts

Fig. 3. Possible functional outcomes of point amino acid mutations in an enzyme with two functional sites (catalytic and a second unrelated Functional Site 2), which are physically separated in terms of the protein’s tertiary mature folded state. Many such mutations may be incompatible with proper folding, triggering cellular responses which recognize and degrade imperfectly folded proteins. In this schematic, a subset of mutations is also noted which are closely localized in either the catalytic site or the second Functional Site 2, where both are compatible with global folding and only adversely influence the local function to which they are proximal. In Category A, catalysis is preserved, but Function 2 is lost, while in the reverse-case Category B, catalysis is ablated by mutation, but the distal Function 2 site remains operational. For a real dual-site moonlighting protein, neither possibility might be possible owing to compromising of global folding, or only one of the two Categories might be feasible in practice.  Also, the ‘clean’ status of the Category A/B dichotomy may not be realized in a real-world situation, where a mutation severely reducing catalytic activity may not totally spare the second Function 2, but still allow enough activity to avoid the phenotypic consequences of complete Function 2 loss. And the reciprocal situation, with a completely null Site 2 mutation accompanied by only partial retention of catalysis, is also formally possible.

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How then do observed patterns of mutations in the clinic with respect to the Lesch-Nyhan SIB phenotype and HPRT levels support or refute the moonlighting hypothesis? If the postulated second ‘neurological’ function of HPRT was associated with a completely distinct region of the protein to the enzyme active site, then it could be possible to find ‘clean’ mutations which knocked out one function while largely preserving the other function. In such circumstances, it would be possible in principle to find germline mutations resulting in enzymatically ablated or minimal HPRT activity, but where no SIB phenotype was manifested (Category B of Fig. 3). Likewise, a mutationally-induced SIB syndrome would be possible where no or little impairment of HPRT enzymatic activity could be found (Fig. 3, Category A).

Certainly mutants of HPRT which retain virtually full catalytic activity in vitro are known. In fact, a triple mutant with replacement of 3 cysteine residues with alanines has been used as an active surrogate of the wild-type enzyme for structural determination, given its greater resistance to oxidation over its natural counterpart. (Keough et al. 2005). It is possible, however, that these mutational changes could have an impact on the hypothetical moonlighting Function 2. These specific mutations have not been described in human patients, and of course cannot be deliberately introduced into the human germline in order to test their effects.

In fact, there have been reports of HPRT mutations in human patients which appear to be consistent with the Category B scenario of Fig. 3 (very low or ablated HPRT without the SIB phenotype). Such findings have been somewhat controversial, since many instances of ‘null’ HPRT activity (especially in relatively early reports) have been discounted by some groups as due to inadequate assay procedures or other experimental shortcomings. Yet it is not at all controversial that clinical HPRT deficiency in itself ranges over a spectrum, from virtually negligible activity even with sensitive assays, to relatively mild impairment. Across this spectrum, cases without the SIB phenotype but HPRT deficiency at various levels has been termed ‘mild’ LND or ‘nsLND’ (non-self-injurious), or grouped as a separate condition (Kelley-Seegmiller syndrome). These considerations aside, if HPRT is solely monofunctional as an enzyme, it would seem a little inconsistent to propose that certain neural cells have an elevated requirement for purine salvage as well as de novo synthesis (which accounts for the LND phenotype), and at the same time insist that even low residual levels of HPRT can allow escape from SIB but not other consequences of salvage deficiency, such as uric acid over-production and deposition.

The moonlighting hypothesis would be consistent with mild LND cases (without SIB) showing distinct patterns of mutations in HPRT, and this indeed has been observed. Nevertheless, it has been noted that the severity spectrum of LND correlates with HPRT enzyme function, where the lowest residual function is seen with the full SIB phenotype. For this to be fully accepted, it is necessary to discount reports of the SIB manifestation in the presence of an HPRT null phenotype, ascribed as noted above to assay problems. Even so, many mutations with global effects would by definition be expected to knock out all or most protein functions, so the correlation of SIB with the lowest enzymatic phenotypes does not exclude moonlighting at all. And a completely ‘clean’ mutation for Category B (Fig. 3) may not exist, where it has such a localized effect that only catalytic activity is ablated without affecting the hypothetical moonlighting function.

But what of the reverse-case Category A scenario of Fig. 3 (normal or effectively functional HPRT in the presence of the SIB phenotype). This is even harder to pin down, but reference to such circumstances has been made in the literature. An obvious problem in this regard, particularly with older reports, is that enzymatic measurements alone are insufficient information. Detection of normal HPRT enzymatic activity in the presence of a SIB phenotype does not answer the question of whether the patient’s corresponding HPRT gene bears any mutations consistent with a Category A (Fig. 3) scenario. Clinical observations that other genetic syndromes can have SIB-like phenotypes (as noted above) are especially pertinent in this regard. Thus, a formal demonstration of the Category A effect would require not only a case of compulsive SIB in association with normal (or near-normal) HPRT levels, but also definitive proof of a coding sequence mutation in the HPRT gene from the same individuals.

Pros and cons for HPRT as a moonlighting protein

If we apply the wisdom of Occam’s Razor, we look first to the simplest explanation that fits the facts, and certainly loss of a single well-characterized activity from the product of the HPRT1 gene is the simplest starting point when attempting to explain the observed Lesch-Nyhan phenotypes. Yet, where the facts demand it, additional complexities may need to be introduced – and biology is notoriously complex in this regard.

In the Table 1 below, some relevant experimental observations are listed, and their interpretations from both the monofunctional (HPRT catalysis as the sole activity of the gene product) and moonlighting interpretations.

Table 1

Table 1. Contrasting interpretations for experimental observations between the moonlighting hypothesis for HPRT (> 1 distinct functions) and the monofunctional catalytic stance. Some of these experimental findings have been discussed above; other relevant examples are also added.

It was of interest that artificially knocking out the murine HPRT gene does not result in a SIB phenotype. Subsequently it was reported that chemical inhibition of the additional salvage enzyme APRT (adenine phosphoribosyl transferase) in HPRT-null mice did result in SIB effects, and this was accordingly cited as an animal LND model. Unfortunately, this was not borne out by additional studies which showed that double APRT-HPRT mouse knock-outs lacked the SIB traits of Lesch-Nyhan victims. In any case, the very different neurological backgrounds between mice and humans could be interpreted as contributing to either moonlighting or monofunctional roles in humans. In the former case, a secondary moonlighting function (lacking in mice) could have been acquired evolutionarily long after the divergence between the common mammalian ancestors of rodents and primates; in the latter monofunctional scenario, an evolutionary role for an increased requirement for guanosine nucleotides in dopaminergic neural signaling (or other pathways) could be likewise proposed as having arisen after the rodent-primate divergence.

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By way of a final note with respect to moonlighting itself, it has been noted that a subset of moonlighting proteins may operate by means of intrinsically unstructured domains, which can assume a specific conformation under the correct physiological circumstances. But from the diversity of known protein moonlighting examples, evidently this does not apply universally. Nevertheless, it is of interest to note that while HPRT does not possess intrinsically disordered domains, it has been shown to undergo exceptionally pronounced conformational changes during its catalytic cycle.  Conceivably, such flexibility could be relevant to its hypothetical second role as a moonlighter during interactions with another biological partner protein or other mediator(s).

In the absence of other information, the possibility of a second moonlighting function for HPRT could be experimentally evaluated with specific searches for its global proteomic interactions in a specific neural cellular background. If HPRT has a defined second role, in principle it should be demonstrable by finding novel partners of it within specific human neural interactomes, especially in comparison with its murine counterparts.

Finally, a global comment in a biopoly(verse) format:

 

As an enzyme, I think that you will find

HPRT has a role well-defined

But its functions may range

Since its loss is quite strange

With the effects that it has on the mind

 

References & Details

(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).

In 1964, William Nyhan and Michael Lesch published a description of a hitherto unrecognized genetic disease…..’ See Lesch & Nyhan (1964). The first encounter of these medical researchers with this condition dates back to 1962, as described by Richard Preston in a New Yorker article in 2007.

The gene encoding HPRT (HPRT1) is situated on the X-chromosome….’     See Fu et al. 2014. Unlike males, females have two copies of the X chromosome, one of which is normally inactivated in each cell as a mechanism for gene expression dosage compensation. Since the inactivation process randomly operates on either X copy in a cell, if an X-linked gene is defective on one copy (heterozygous), on average 50% of cells will contain a functional X chromosome copy correctly expressing the gene of interest. This will reduce the overall expression level, and in the case of HPRT1 can be associated with an increased propensity for gout in affected females, depending on the specific mutation involved, or other factors. Sufficient HPRT expression from a single functional X chromosome HPRT1 copy is, however, sufficient to prevent the neurological symptoms of Lesch-Nyhan disease from arising.

‘……self-injurious behavior (SIB) can take the form of severe lip and finger biting, or other forms of self-damage…..’     See Preston 2007; Fu et al. 2014.

Human HPRT is a protein of 217 amino acid residues….’     The N-terminal methionine residue is naturally cleaved off, such that numbering of the HPRT sequence begins with the N-terminal alanine residue (Keough et al. 2005).

Structure of a monomer of human HPRT in complex with guanosine monophosphate ’ (Fig. 2).      It has been found that HPRT protein in the absence of either its substrates or products (as in Fig. 2, with GMP) is unstable in vitro. The free enzyme was eventually successfully crystallized and structurally resolved by the use of a triple mutant with 3 of the 4 natural cysteine residues replaced with alanines, a manipulation which did not affect its enzymatic behavior (Keough et al. 2005).

This image has been generated from Protein Databank entry 1HMP….’ (Fig. 2).      The structure was derived by Eads et al. 1994. The image itself was generated from the Protein Databank 1HMP entry with Protein Workshop software (Moreland et al. 2005).

Most organisms possess both synthetic approaches towards purines in their biochemical repertoires….’      A notable exception to this guideline are certain protozoan parasites, which have lost the de novo purine synthetic pathway in favor of exploiting freely available host purines through their own salvage pathways. See el Kouni 2003.

The de novo synthesis of purines involves numerous enzymatic steps….’      For more detail, see Biochemistry 5th Edition, Berg et al. 2002.

‘…..multienzyme complexes termed ‘purinosomes’….’      See Pedley & Benkovic 2017.

This kind of functional separation [between distinct protein sites associated with specific functions] ……’  See a previous post with specific reference to the Large T protein of SV40 virus, which has at least seven different sites performing different functions.

‘……the possibility of a moonlighting role for HPRT in itself it is not an original suggestion…..’      See Ceballos-Picot et al. 2009, for a mention of conceivable HPRT moonlighting in the Discussion of this paper.

‘…….the notion of biological parsimony……’      See previous posts 21 April 2015; 24 August 2015; 21 February 2016; and 28 August 2016.

‘……perturbations in dopamine-mediated (dopaminergic) neural pathways have been reported in LND….’      See (for example) Bell et al. 2016.   ‘….and associated experimental cellular models.’      See Kang et al. 2013, in a study of the effects of HPRT knock-down on developmental pathways of murine embryonal stem cells (ESCs). Once again, evidence for a role for HPRT in dopaminergic signaling / neural development does not preclude its functioning in a moonlighting role distinct from catalysis, as the latter could be directly connected with a neural signaling / developmental pathway in a manner that has no direct connection with the purine salvage catalytic role. In any case, whatever the effects of HPRT deficiency in the murine ESC system, complete ablation of HPRT in mice does not result in a SIB phenotype, as noted below.

A clinical observation consistent with the above-noted role of dopaminergic signaling in LND is a certain level of responsiveness of LND patients to D1 dopamine receptor antagonists in reducing SIB. With respect to this, see Khasnavis et al. 2016.

‘…….treatment of LND patients with an inhibitor of uric acid formation….’      The inhibitor is allopurinol, which blocks the activity of the enzyme xanthine oxidase. See Hitchings 1975; De Antonio et al. 2002.

‘……If salvage deficiency meant that neurones cannot maintain sufficient levels of purine nucleotides, it should be quantifiable…..’      See Bell et al. 2016;  Fu et al. 2015.

‘…..up-regulation of de novo purine synthesis, evidenced by high purinosome levels in Lesch-Nyhan cells….’   /   ‘ ….. it has been suggested that de novo synthesis may be nevertheless unable to cope with certain circumstances of unusually high purine demand….’ See Fu et al. 2015.

‘….in the absence of salvage pathways may lead to the accumulation of neurotoxic by-products…..’      See Sidi & Mitchell 1985.

‘…little of no bearing on effective function.’      Here “little” indicates a loss of efficiency that might have negatively selectable consequences for an organism in natural circumstances, but which would have small effects on human beings in most present day life situations. Of course, the flip-side is the possibility of a mutation with increased efficiency, which might under natural conditions become itself positively selected for.

‘…….a triple mutant with replacement of 3 cysteine residues with alanines has been used as an active surrogate of the wild-type enzyme for structural determination……’     See  Keough et al. 2005.

‘….clinical HPRT deficiency in itself ranges over a spectrum….’  /  ‘……mild LND cases (without SIB) showing distinct patterns of mutations in HPRT…..’   /  ‘…..the severity spectrum of LND correlates with HPRT enzyme function…..’     See Jinnah et al. 2010.

‘….reports of HPRT mutations in human patients which appear to be consistent with the Category B scenario of Fig. 3 (very low or ablated HPRT without the SIB phenotype).’      See Rijksen et al. 1981 (with discussion of other such examples); Bayat et al. 2014; Bell et al. 2016.

‘……many instances of ‘null’ HPRT activity (especially in relatively early reports) have been discounted by some groups as due to inadequate assay procedures or other experimental shortcomings.’     See Fu et al. 2014.

‘…..a separate condition (Kelley-Seegmiller syndrome).’     See Kelley et al. 1969.

‘……reference to such circumstances [the Category A scenario of Fig. 3 (normal or effectively functional HPRT in the presence of the SIB phenotype] has been made in the literature.’     See Rijksen et al. 1981, who cite Etienne et al. 1973; Encéphalopathie hypèruricosurique avec auto-mutilations. Rev Rhum Mal 40: 265-270.

Table 1. ‘…….artificially knocking out the murine HPRT gene did not result in a SIB phenotype…….’  See Finger et al. 1988.

‘…chemical inhibition of the additional salvage enzyme APRT (adenine phosphoribosyl transferase) in HPRT-null mice did result in SIB effects…..’     See Wu & Melton 1993.

‘….additional studies which showed that double APRT-HPRT mouse knock-outs lacked the SIB traits of Lesch-Nyhan victims.’     See Engle et al. 1996.

‘….a subset of moonlighting proteins may operate by means of intrinsically unstructured domains….’     See Tompa et al., 2005.

‘…..the diversity of known protein moonlighting examples….’      See the moonlighting protein database, as of 2018, and its associated publication (Chen et al. 2018). As an additional note, while this database holds no entry for HPRT, it does contain a representative of a different nucleotide salvage pathway, in the form of human thymidine phosphorylase, which acts both in the salvage-related production of thymidine monophosphate, and with the unrelated function of platelet-derived endothelial cell growth factor.

‘…..HPRT ……. has been shown to undergo exceptionally pronounced conformational changes during its catalytic cycle.’      See Keough et al. 2005.

‘….a second moonlighting function for HPRT could be experimentally evaluated with specific searches for its global proteomic interactions in a neural cellular background.’     These include high-throughput 2-hybrid assays or mass-spectroscopic approaches.

Next post: April-May.

 

 

 

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