Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP)

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase is a hydrolase enzyme which is known to be expressed in the liver, kidney, and at moderate levels in the brain^[1]. It belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases with hydrolyze a wide variety of substrates^[2]. LHPP, specifically, hydrolyzes both oxygen-phosphorous bonds in inorganic phosphate and nitrogen-phosphorous bonds in 6-phospholysine, 3-phosphohistidine, and imidodiphosphate. LHPP has been of particular interest to researchers in recent years due to its hypothesized function as a tumor suppressor and thus its role in various cancers^[3].

The HAD Superfamily

The haloacid dehalogenase superfamily contains over 79,000 unique sequences of enzymes and is largely made up of enzymes that catalyze phosphoryl transfer^[1]. (phosphate monoester hydrolases) make up the majority of these enzymes at ~79%, with many of the rest being (phosphoanhydride hydrolases)^[2]. While many members of the enzyme family are found predominantly in prokaryotes, 183 human HAD enzymes have been identified, with at least 40 HAD-type phosphatases. This ancient group of enzymes has evolved over time to dephosphorylate a wide variety of substituents including carbohydrates, lipids, DNA, and various amino acid-phosphorylated proteins in humans, though many target small metabolites in intermediary metabolic reactions. These enzyme were originally thought to carry out simple regulatory tasks, but recent research has shown that some of these enzymes play roles in various genetic disorders^[1].

Sequentially, there is very low similarity across the HAD phosphatases, so members of the family are instead identified using alignments of amino acid sequences that are based on the presence of four short signature motifs that contain conserved catalytic residues present in HAD enzymes. Another similarity between the HAD phosphatase superfamily is that all the enzymes share the same active core structural arrangement, consisting of catalytic machinery residues positioned in a Rossmann fold. This super-secondary structure is characterized by an alternating motif of repeating β-α units arranged in three stacked α/β sandwiches. The Rossmann fold of HAD phosphatases also contains three unique structural signatures including the squiggle, flap, and cap domains. These domains allow HAD phosphatases to form different conformational states as well as influence substrate specificity^[2].

HAD Phosphatases: Mechanism & Structure

The catalysis mechanism of HAD phosphatases is unique in comparison to other phosphatases and requires the use of an aspartate residue in the . This residue facilitates a nucleophilic attack and also contributes to these enzymes' lack of sensitivity to common phosphatase inhibitors. This attack is carried out in a two-step phosphoaspartyl transferase mechanism. As previously mentioned, the aspartate residue initiates the nucleophilic attack on the substrate's phosphoryl group, displacing the substrate's leaving group and forming a phosphoaspartyl enzyme intermediate. In the second step, a water molecule initiates a nucleophilic attack on the previously formed intermediate, releasing free phosphate and regenerating the aspartate catalyst. There is also a second Asp residue, designate Asp + 2, which functions as a general acid/base to protonate the leaving group of the substrate in the first reaction and deprotonate the water molecule in the second reaction^[2].

It is also important to mention that all HAD phosphoaspartyl transferases require as an obligatory cofactor. This cofactor has multiple functions, including positioning of the substrate phosphoryl group in relation to the Asp nucleophile, providing electrostatic stabilization and charge neutralization in the transition state^[2].

LHPP-Specific Mechanisms & Structure

LHPP is a phosphoramidase that forms a homodimer in solution and is involved in the cleavage of P-N and O-P bonds. This protein contains a Ser residue where other members of the HAD family contain Asp or Thr residues. It also contains a Ser + 2 residue, which is unique to mammalian HAD-type hydrolases. LHPP is a capped HAD phosphatase (meaning it contains a cap domain) with a C2a-type cap domain. Cap domains of HAD phosphatases are integral to controlling access to the active site via shielding and determining substrate specificity. Some capped HAD phosphatases, like LHPP, are able to act on in addition to their other functions. This is particularly interesting due to the occluded nature of the active sites of phosphoproteins, making them difficult to access. LHPP, along with other C2a-capped HAD phosphatases, has been shown to act on serine-, tyrosine-, and histidine-phosphorylated proteins. The subcellular localization of LHPP is currently unknown, but proposed locations are the nucleus and cytosol. The enzyme is also predicted to interact with ATP synthase subunits and theorized to play a role in oxidative phosphorylation^[1].

Role in Disease

LHPP has been implicated as a risk factor for major depressive disorder and various cancers in multiple studies, with a range of mechanisms at play^[1]. Different research methods have been used to investigate this topic including genetic sequencing, rs-fMRI scans, cell culturing, various assays, and mass spectrometry^[3]. Much of the research on this enzyme, particularly its involvement in cancer, is relatively new and ongoing.

Major Depressive Disorder

On single-nucleotide polymorphism (SNP) at the LHPP gene (rs35936514) has been identified as having a potential role in Major Depressive Disorder (MDD) in genome-wide association studies. A study of 160 patients with a range of genotypes (a CC group homozygous for the C allele and a CT/TT T-carrier group carrying the high-risk T-allele) was assessed by brain activity using the amplitudes of low-frequency fluctuations (ALFF). Patients with MDD in the T-carrier group showed increased ALFF in the left superior temporal gyrus, indicating that this genotype may influence regional brain activity^[4]. Another SNAP associated with serotonin receptor 1A-1019C (G genotype, present in Utah and Ashkenazi populations) has also been implicated in enhanced expression of the HTR1a autoreceptor which has been linked to reduced serotonergic neurotransmission, considered to be one of the primary defects in depression. Other studies have more widely implicated the LHPP locus as playing a role in MDD^[1].

Cancer

Different LHPP polymorphisms have been identified as functionally relevant in a wide variety of cancers including pharyngeal cancer, B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and testicular germ cell tumors. The LHPP SNP rs201982221 was identified, along with 7 other novel risk loci, to increase susceptibility for oral cavity and pharyngeal cancers. LHPP polymorphism rs35837782 was identified as a common risk locus for BCP-ALL, however this SNP did not appear to influence patient outcome. And intronic LHPP SNP rs61408740 was associated with testicular germ cell tumor susceptibility^[1].

While LHPP SNPs have been linked to an increased risk for certain cancers, the enzyme has been shown to function as a tumor suppressor and contribute to other cancers in this way. Although histidine phosphorylation is poorly characterized, this method of post-translational modification of proteins has been shown to be oncogenic when disregulated^[1]. Downregulated expression of the putative histidine phosphatase LHPP was found specifically in tumor cells in one study of hepatocellular carcinoma. This downregulation appears to lead to an upregulation of global histidine phosphorylation with increased tumor severity and a decrease in overall survival^[3]. Down-regulation of LHPP was also shown to influence cervical cancer in another study, which also looked at LHPP over-expression. Over-expression of the enzyme appeared to reduce cancer cell proliferation, migration, and invasion and was associated with a change in metastasis signaling pathways. It was also shown to induce apoptosis in human cervical cancer cells and block AKT activation, all of which are considered anti-cervical cancer effects and could prove useful in developing new therapeutic strategies^[5]. LHPP regulation of colorectal cancer cells via the PI3K/AKT pathway has also been studied^[6].

Thyroid Diseases

One study has implicated increased nuclear expression of LHPP in association with hyperthyroidism, though it is unclear is these results have since been replicated. This study looked at the intranuclear expression of LHPP thyrocytes and showed enhanced expression in Graves' disease, though it concluded that LHPP expression may not actually be regulated by disease-derived serum factors^[7].

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7} Gohla A. Do metabolic HAD phosphatases moonlight as protein phosphatases? Biochim Biophys Acta Mol Cell Res. 2019 Jan;1866(1):153-166. doi:, 10.1016/j.bbamcr.2018.07.007. Epub 2018 Jul 18. PMID:30030002 doi:http://dx.doi.org/10.1016/j.bbamcr.2018.07.007
2. ↑ ^{2.0 2.1 2.2 2.3 2.4} Seifried A, Schultz J, Gohla A. Human HAD phosphatases: structure, mechanism, and roles in health and disease. FEBS J. 2013 Jan;280(2):549-71. doi: 10.1111/j.1742-4658.2012.08633.x. Epub 2012 , Jun 13. PMID:22607316 doi:http://dx.doi.org/10.1111/j.1742-4658.2012.08633.x
3. ↑ ^{3.0 3.1 3.2} Hindupur SK, Colombi M, Fuhs SR, Matter MS, Guri Y, Adam K, Cornu M, Piscuoglio S, Ng CKY, Betz C, Liko D, Quagliata L, Moes S, Jenoe P, Terracciano LM, Heim MH, Hunter T, Hall MN. The protein histidine phosphatase LHPP is a tumour suppressor. Nature. 2018 Mar 29;555(7698):678-682. doi: 10.1038/nature26140. Epub 2018 Mar, 21. PMID:29562234 doi:http://dx.doi.org/10.1038/nature26140
4. ↑ Cui L, Gong X, Tang Y, Kong L, Chang M, Geng H, Xu K, Wang F. Relationship between the LHPP Gene Polymorphism and Resting-State Brain Activity in Major Depressive Disorder. Neural Plast. 2016;2016:9162590. doi: 10.1155/2016/9162590. Epub 2016 Oct 23. PMID:27843651 doi:http://dx.doi.org/10.1155/2016/9162590
5. ↑ Zheng J, Dai X, Chen H, Fang C, Chen J, Sun L. Down-regulation of LHPP in cervical cancer influences cell proliferation, metastasis and apoptosis by modulating AKT. Biochem Biophys Res Commun. 2018 Sep 5;503(2):1108-1114. doi:, 10.1016/j.bbrc.2018.06.127. Epub 2018 Aug 2. PMID:29944886 doi:http://dx.doi.org/10.1016/j.bbrc.2018.06.127
6. ↑ Hou B, Li W, Li J, Ma J, Xia P, Liu Z, Zeng Q, Zhang X, Chang D. Tumor suppressor LHPP regulates the proliferation of colorectal cancer cells via the PI3K/AKT pathway. Oncol Rep. 2020 Feb;43(2):536-548. doi: 10.3892/or.2019.7442. Epub 2019 Dec 20. PMID:31894339 doi:http://dx.doi.org/10.3892/or.2019.7442
7. ↑ Koike E, Toda S, Yokoi F, Izuhara K, Koike N, Itoh K, Miyazaki K, Sugihara H. Expression of new human inorganic pyrophosphatase in thyroid diseases: its intimate association with hyperthyroidism. Biochem Biophys Res Commun. 2006 Mar 17;341(3):691-6. doi:, 10.1016/j.bbrc.2006.01.016. Epub 2006 Jan 18. PMID:16430861 doi:http://dx.doi.org/10.1016/j.bbrc.2006.01.016