Inteins and Exteins- The Story of Protein Splicing Machinery

Any undergraduate or a graduate student in the field of advanced biology might be familiar with the terms introns, exons and mRNA splicing. But if someone ask them about inteins, exteins and protein splicing, less likely that they will be knowing. Hence, for this week I thought to discuss about these special processes that occur virtually in all three domains of life.

What are Inteins and Exteins?

Inteins were first reported in 1990 while working on vacoular proton ATPase in Saccharomyces cerevisiae. As many as more than 600 putative genes for inteins (word inteins means both inteins and exteins) have been identified till date. The acronym for intein and extein is internal protein segment and external protein. Taken together these perform a form of “post translational modification” where a part of protein get excised itself (i.e. intein) from the translated sequence and ligates the flanking ends (C and N terminus) to form a mature protein (i.e. extein).

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Fig 1:  The protein splicing mediated by inteins and exteins

Image adapted from Elleuche & Pöggeler (2010), Applied Microbiology and Biotechnology

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Fig 2: A comparison between protein slicing and RNA splicing

Image source: The Inteins database at http://www.inteins.com

The portion of unspliced extein on the N- and C-terminal sides of the intein are called the N-extein and the C-extein. Amino acid sequence studies has shown that the exteins do not possess any catalytic activity and are vary from one proteins to other. Whereas the structure and sequence of the roughly ~130 amino acid inteins is highly conserved with the following splice junction residues

(1) a Ser/Thr/Cys at the intein’s N-terminus; and

(2 and 3) a His–Asn/Gln dipeptide at the intein’s C-terminus; which is immediately followed by

(4) a Ser/Thr/Cys at the N-terminus of the C-extein

Reactions

Fig 3: Series of reactions catalyzed by inteins to splice themselves out of a polypeptide chain. 

Image source: The Inteins database at http://www.inteins.com

The reaction catalyzed by inteins and exteins does not require ATP and is an autocatalytic reaction!

What are the functions of Inteins and Exteins?

The biological function of presence of inteins and exteins is enigmatic, but most of the proteins having these sequences are actually homing endonucleases (HED) that involve in gene conversion. These HEDs create a break in genes that are homologous to corresponding extein but which lack intein and then initiate a site specific recombination by activating double stranded break repair (DSB) pathway. Since, the intein containing gene is only likely gene containing extein like sequence, the intein gene is copied on to the homologous chromosome. Thus the primary function thought to be evolved as a mechanism of propagating the so called junk DNA or parasitic DNA. A careful observation reveals that the relation between intein’s protease activity and endonuclease activity exhibit a symbiotic relationship. The protease activity excises the intein sequence whereas the endonuclease assures the transposition of the intein gene.

 

Application of Inteins in Biotechnology

Though inteins do not carry out any other known function besides their selfish propagation of DNA, their properties are cleverly exploited in biotechnology based applications. The first and foremost property that makes them interesting tool is the efficiency in splicing the proteins and generate a properly folded protein. Inteins can be used to perform tagless proteins purification. In this process a point mutation will be introduced in the conserved  splicing motifs (discussed above) along with a purifying tag attached to intein sequence which will be excised after purification by just changing the pH of the medium. The special property of these proteins also found a place in gene therapy trials in mammalian cell cultures for treating mitochondrial disorders experimentally. One major obstacle in the field of gene therapy for mitochondrial disorders is the hydrophobicity of mt proteins hindering the import through the Mito

Fig 4: A method by which inteins might improve the mitochondrial import of highly hydrophobic proteins (a). (b) The hydrophobic domains are moved further apart, allowing their import to be more cotranslational. Image adapted from: Grey, Trends in Biotechnology (2000)

mitochondrial double membrane. The problem can be solved by placing a intein sequence between a coding region of resident protein thus making it less hydropohobic and enhacing transport. Once imported, the intein sequence will be spontaneously excised forming a mature protein. The couple of applications mentioned in this post is just  tip of an iceberg. Inteins will play a key role in biological pharmaceuticals in near future.

REFERENCES: 

  • Donald Voet and Zudith Voet (2012), Biochemistry, 4th edition, John Wiley Publication.
  • Skander Elleuche & Stefanie Pöggeler (2010). Inteins, valuable genetic elements in molecular biology and biotechnology. Applied Microbiology and Biotechnology 87:479–489.
  • Neel H. Shaha and Tom W. Muira (2014). Inteins: Nature’s Gift to Protein Chemists. Chemical Science  5: 446–461.
  • Aubrey D.N.J de Grey (2000).  Mitochondrial gene therapy: an arena for the biomedical use of inteins. Trends in biotechnology 18:394–399.