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14-3-3 Protein Analysis | Essay

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INTRODUCTION

14-3-3 represents a family of highly conserved, 28-33 kDa, acidic dimeric proteins. [B, H1] These proteins were first identified and isolated by Moore and Perez during their systematic study of protein fractions from mammalian brain tissue. [B1] Their nomenclature reflects the system used by the researchers to classify the various protein fractions, based on their migration pattern on starch gel electrophoresis and two-dimensional DEAE chromatography. [D1]

Initially it was thought that the occurrence of these proteins was confined to neuronal tissue, where they were found to be present in concentrations as high as 13.3 μg/ml. [B19] However, subsequent studies showed that they are in fact widely distributed and ubiquitously expressed in most mammalian tissues. [B]

Apart from mammalian tissue, orthologous proteins with a high degree of similarity have also been identified in other organisms including plants, yeast, insects and amphibians. [B] Interestingly, though no prokaryotic ancestor has been identified for the 14-3-3 gene, eukaryotes are known to express multiple isotypes, encoded by highly similar gene sequences. [A, G13] Drosophila and yeast each contain two 14-3-3 genes, Arabidopsis species have 15, while the human genome contains seven closely related 14-3-3 genes. [G13-G16]

These genes encode the seven different 14-3-3 isotypes found in human cells – namely β, γ, σ, ε, η, τ, and ζ. Two additional isoforms – α and δ, represent the phosphorylated varieties of β and ζ respectively. [D7]

By virtue of their conserved sequences, the different 14-3-3 isoforms display a similar tertiary structure. Crystallographic studies show that each subunit is made up of nine anti-parallel α helices, which are able to self-assemble into dimers [F1]; with four of the nine helices participating in this process of dimerization. [A] While certain isoforms such as σ and γ show propensity towards the formation of homodimers, other isoforms like ε prefer to heterodimerize. [H] Structural analyses have also revealed that each monomer contains an amphipathic concave channel through which it interacts with its target proteins. [F13] Since each of the subunits of the dimer contain an independent ligand-binding groove, these proteins can interact with two different binding sites present on the same or different target proteins [H3]

The targets of 14-3-3 proteins were first studied by Muslin et. al. in 1995, who determined that these proteins interact with phosphorylated serine-containing binding partners in a sequence specific manner. [A2] Thus, this came about to be the first family of proteins discovered to have an affinity for phosphoserine-specific targets. More specifically, Yaffe et. al. in 1997 identified two putative high-affinity motifs, bearing the sequences RSXpSXP and RXXXpSXP, where pS stands for phosphoserine, R stands for Arginine, P is proline and X is any amino acid. [] These were found to be the consensus sequences for 14-3-3 binding, and were called the mode I and mode II binding motifs respectively. [A21] However not all the 14-3-3 binding partners conform to these motifs and neither are all 14-3-3-target interactions found to be phosphorylation-dependent. [H]

Though, a small percentage of 14-3-3 targets do not conform, in general it can be said that, proteins that interact with the 14-3-3 family are usually found to be globular proteins that contain either the mode I or mode II binding motifs within an unstructured region of the peptide. [PG] Interestingly, despite the fact that the different isotypes share a similar structure and show complete conservation of sequence in the ligand-binding region, not all the isotypes of 14-3-3 bind equivalently to their ligands in vivo and the pathways in which the individual isoforms participate have diverged considerably. This isoform specificity cannot be explained solely on the basis of 14-3-3 binding to the consensus sequence. [A, PA] It has hence been speculated that this isoform-specificity may be attributed either to differences in subcellular localization and/or tissue-specific transcriptional regulation rather than inherent differences in their ligand-binding ability. [D]

Consequently, the various isoforms of the protein are found to bind different ligands and hence influence distinct cellular pathways and processes. Some of the ligands postulated on the basis of database searches include Raf1, CDC25C, polyoma middle T antigen, BAD, 5’AMP Kinase and PLC γ. [PA] Studies conducted in the late 1990’s demonstrated the ability of 14-3-3 proteins to interact with a number of different protein phosphatases and kinases in the cell, thus influencing multiple signalling pathways within the cell. [D]

The mechanism of action of the 14-3-3 family of proteins is based on their ability to alter the stability, the catalytic activity or the subcellular localization of their targets. [H] 14-3-3 dimers being highly rigid, they are able to induce conformational changes in their target proteins as well as generate steric hindrance upon binding. Together, these forces can prevent molecular interactions by modulating the accessibility of the ligand to enzymes; and they can also serve to expose or hide localization motifs such as NES and NLS. [H]

The first cellular activity to be attributed to 14-3-3 was that of an activator of tryptophan and tyrosine hydroxylases, rate-limiting enzymes that regulate the biosynthesis of catecholamine and serotonin neurotransmitter, as reported by Ichimura and co-workers. [B20] Subsequently it was discovered that 14-3-3 proteins could regulate the activity of signal transduction molecule protein kinase C. [D3,D4] This and other findings led to the implication that 14-3-3 proteins could act as novel chaperone proteins that are able to modulate the interactions between the different constituents of signal transduction pathways. [D5]

Today it is known that 14-3-3 protein dimers are able to interact with a wide array of proteins within the cell including signalling molecules, apoptosis factors, tumor supressors, transcription factors, biosynthetic enzymes as well as cytoskeletal proteins; as a result of which they play crucial roles in the regulation of multiple cellular process such as the onset of cellular differentiation, and senescence, DNA repair and the maintenance of cell cycle check point, co-ordination of motility and adhesion and the prevention of apoptosis [A,H]

REVIEW OF LITERATURE

14-3-3 proteins are crucial in the regulation of many cellular functions. An implication of their involvement in this multitude of processes is that any mutation, loss of regulation or altered expression of these proteins can lead them to be associated with major diseases. Through their involvement in the regulation of various tumor suppressor genes and oncogenes, 14-3-3 proteins are thought to be potentially involved in cancerous transformation and the development of malignancies. [Z11] They are also known to be associated with neurodegenerative conditions such as Alzheimer’s disease, ataxia and Parkinson’s disease. [MT1, MT2, MT3, MT4]

The causes of these diseases are poorly understood and hence this family of proteins has been the focus of study of several research groups around the world, leading to numerous publications investigating all the different aspects. This section summarizes the literature elucidating the properties and functions of 14-3-3 proteins that have helped shape the rationale behind this project. A special focus is maintained on current literature that highlights role of 14-3-3 γ in the cell cycle and in desmosome assembly, as well as its recently discovered ATPase activity.

Human 14-3-3 isoforms

The members of the 14-3-3 family are among the most abundant proteins in the cell. It has been established that owing to their interaction with diverse targets, 14-3-3 proteins are known to be involved in a multitude of processes, including the control of gene transcription, metabolism, cell cycle regulation, and apoptosis. [C] However, not all 14-3-3 proteins carry out the same functions. The table below provides an overview of the properties of the various human 14-3-3 isoforms:

14-3-3 isoform

Official symbol

Official full name

Chrom-osome location

No. of amino acids

Mol.Wt. (kDa)

Interactions, functions and disorders

14-3-3 β

YWHAB

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta

20 q13.1

246

27.95

  • shown to interact with CDC25 phosphatases and RAF1
  • may play a role in relaying mitogenic signaling to the cell cycle machinery

14-3-3 γ

YWHAG

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma

7 q11.23

247

28.12

  • interacts with RAF1, CDC25C and protein kinase C
  • induced by growth factors in human vascular smooth muscle cells
  • also expressed significantly in heart and skeletal muscles

14-3-3 σ

YWHAS, SFN

Stratifin

1 p36.11

248

27.64

  • known to interact with PLK4, ERRFI1, MARK3, JUB
  • expressed in epithelial cells

14-3-3 ε

YWHAE

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon

17 p13.3

255

29.03

  • interacts with RAF1, CDC25 phosphatases, and IRS1 proteins
  • plays a role in pathways related to signal transduction, cell division and regulation of insulin sensitivity
  • implicated in the pathogenesis of small-cell lung cancer

14-3-3 η

YWHAH

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta

22 q12.3

246

28.09

  • contains a repeating sequence, 7 bp in length, in its 5' UTR
  • changes in the number of repeats are associated with conditions such as psychotic bipolar disorder and schizophrenia

14-3-3 τ/θ

YWHAQ

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta

2 p25.1

245

27.63

  • found to be over-expressed in patients with amyotrophic lateral sclerosis
  • also expressed in T cells

14-3-3 ζ

YWHAZ

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta

8 q23.1

245

27.61

  • interacts with IRS1 protein
  • may play a role in regulating insulin sensitivity

14-3-3 γ

14-3-3 γ is an adapter protein that is known to be involved in the regulation of a wide range of signalling pathways, both general and specialized. This protein is encoded by a gene designated YWHAG, which was mapped to the chromosome 7q11.23 by Horie et. al. in 1999 [Z2] A study of its binding partners in a yeast two-hybrid study revealed that 14-3-3 γ may have as many as 130 potential ligands. [PG 4]

There are reports which link the overexpression of 14-3-3 γ and its high titres in the cerebrospinal fluid (CSF) with certain neurodegenerative disorders such as Down syndrome and Alzheimer’s. [A129, A131]

This protein is also thought to to be involved in the process of carcinogenesis. Although 14-3-3 σ is the isoform that has been most extensively studied for its association with human cancers, there are also several ongoing studies which attempt to establish a correlation between 14-3-3 γ expression and the development of tumours. This includes the work of Jieqiang Lv. et. al. which was able to demonstrate through proteomic analysis that a reduced expression of 14-3-3 γ is observed in patients with uterine tumours. [Z10]

The role of 14-3-3 γ in cancer can be better appreciated by understanding its role in the cell cycle and its regulation.

Role of 14-3-3 γ in the cell cycle

14-3-3 proteins play a major role in the regulation of cell cycle through the interaction with various cell cycle proteins. The main targets for 14-3-3 regulation are the Cdc25 proteins. Cdc25 is a family of protein phosphatases comprising the members Cdc25A, Cdc25B and Cdc25C, which are active during different phases of the cell cycle. Cdc25A participates in the regulation of G1/S transition, whereas Cdc25B and Cdc25C regulate G2/M transition. [z3]

Cdc25c has been specifically implicated in the activation of the CDK1-cyclinB1 complex by removing inhibitory phosphorylations at T14 and Y15, which advances the cell cycle from G2 to M phase. The overexpression of this protein has been seen to result in mitotic catastrophe in the cell due to premature mitosis. [E28] Therefore the expression and function of Cdc25C needs to be tightly regulated; and 14-3-3 γ plays a crucial role in this regulation. [E29]

During the interphase, 14-3-3 proteins bind to Cdc25C and sequester it in the cytoplasm by occluding the NLS, preventing it from accessing the CDK1-cyclinB1 complex in the nucleus and thus in essence inactivating it. [E28] Dalal et. al. showed that though all the 14-3-3 proteins were able to carry out this function in vitro, only 14-3-3 γ and ε were found to bind to and thus inhibit Cdc25C in vivo. []

This binding however was found to require the phosphorylation of a specific serine residue at position 216 in human cells, usually carried out by TAK1 and other similar kinases. [E30, z4] Cdc25C is also known to be a target of the DNA replication and DNA damage checkpoints that exert their effects through the phosphorylation of S216 by kinases like Chk1 and Chk2. [z5, z6, z7] This phosphorylation thus generates a high-affinity motif that is able to bind to 14-3-3 proteins present in the cytosol.

A schematic representation of this process is provided in the figure below.

In the absence of 14-3-3 proteins, this regulation of Cdc25C is disrupted and a result, cells lose their ability to stop cycling upon loss of integrity of their DNA. To this effect, a study conducted by Hosing et. al. in 2008 showed that cells require 14-3-3 γ in order to maintain their ability to arrest cells in the S phase and the G2 phase checkpoint. [] This was proven by generating 14-3-3 γ knockdown cells, and studying their response to DNA damage. It was found that these cells were unable to arrest in G2, thus leading to an increase in premature chromatin condensation (PCC) as compared to the vector control. [] This was the first report suggesting that 14-3-3 γ modulates the cell cycle checkpoint response, through its negative regulation of cdc25C function.

Role of 14-3-3 γ in desmosome assembly

14-3-3 isoforms were also not known to have any role to play in the assembly of desmosomes, until a serendipitous observation led to a study conducted by Sehgal et. al. This study demonstrated for the first time that 14-3-3 γ is required for desmosome formation. [] They showed that 14-3-3 γ is able to form complexes with the desmosomal proteins plakoglobin, desmoplakin and plakophilin 3. Their work led to the conclusion that 14-3-3 γ is essential for the recruitment of plakoglobin to the cell border; and that the loss of 14-3-3 γ leads to a decrease in desmosome formation. This translates to defective cell-cell adhesion in HCT 116 cells and sterility in male mice when this loss is manifested in the testis. [] These results are in agreement with the fact that 14-3-3 γ plays a role in the transport of proteins from the Golgi complex to the cell border, as previously reported by Valente et al. in 2012. [Z1]

ATPase activity of 14-3-3 proteins

The notion that 14-3-3 proteins may possess an ATPase function evolved through a series of studies. The first of these was reported in a paper in 1993 by Hachiya et. al., who had isolated a protein from the rat liver cell cytosol, called the mitochondrial import stimulation factor (MSF). [] This factor was found to modulate the conformation of aggregated proteins and stimulate their import into the mitochondria in an ATP-dependent manner, much like the Hsp70 chaperone molecule. Ensuing studies by the same group led to the cloning of cDNA sequences encoding the large and small rat MSF subunits; and based on these cDNA sequences, it was deduced that the MSF peptides belong to the 14-3-3 family. [] This was thus the first time the ability to hydrolyse ATP was attributed to a 14-3-3 protein.

In 1997, Yano et. al. probed further and asked whether the 14-3-3 family of proteins were also capable of the reverse reaction i.e. ATP synthesis; and found that native 14-3-3 proteins isolated from human lymphoblastoma cells as well as recombinant 14-3-3 τ exhibited the ability to generate ATP from ADP. [] This activity resembled that of the enzyme nucleoside diphosphate-kinase. It was then speculated that ATP synthesis and hydrolysis may play a role in regulating the interaction of 14-3-3 proteins with their substrates. Subsequently, in 2006, Yano et. al. also showed that 14-3-3 ζ acts as a molecular chaperone under heat shock conditions, dissolving thermally-aggregated proteins, also in an ATP-dependent manner. [] This stress-related function is said to differ from the role of MSF as previously described, since in that case, the protein activity is limited to the transport of newly synthesized proteins into the mitochondria. This finding thus represented another part of the puzzle that is the multi-functional nature of 14-3-3 proteins.

Despite these reports however, the ATPase activity of 14-3-3 proteins had not commanded much attention from researchers, until recently Ramteke et. al. in 2014 demonstrated unequivocally, using 14-3-3 ζ as a model, that these proteins possess an intrinsic ATP-hydrolysing function. [] They also studied the putative residues essential to this activity and found that in their model, the mutation of the Asp at residue 124 to Ala (D124A) resulted in a significant increase in the rate of ATP hydrolysis, while the mutation of Arg 55 to Ala caused a partial loss of function.

ATPase activity of 14-3-3 γ

As a part of the same study, Ramteke et. al. decided to test whether the other 14-3-3 isoforms also displayed similar properties; and they found that with the exception of 14-3-3 σ, all the other isoforms, including 14-3-3 γ, showed intrinsic ATPase activity. [] Interestingly, it was found that in case of the γ isoform, the mutation of the conserved Asp, found at position 129 to Ala (D129A) led to an over two-fold gain in ATPase function, reflecting that the binding of this mutant to ATP is probably either more energetically or sterically favourable. [] The authors of this paper were however unable to comment on the functional relevance of the ATPase activity of 14-3-3 proteins. These results however provide an impetus to further studies on physiological significance of ATP hydrolysis, with respect to individual 14-3-3 isoforms.


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