‘Nature’s escape artists’

Authors explore the various functions
and applications of intein-mediated protein splicing

Cover of the JBC thematic minireview series on prokaryotic protein phosphorylation

Post-translational protein splicing occurs when intervening intein polypeptides excise themselves from larger precursor proteins and ligate their surrounding polypeptides, known as exteins. This is accomplished by a multistep enzymatic reaction mediated by the intein. Sometimes called “nature’s escape artists,” inteins are proteins that have been found in microorganisms from all domains of life.
Even though the first intein sequence was published 25 years ago, details of the splicing mechanism are just beginning to be elucidated. Inteins have fascinated scientists for years, and they have been shown to have a wide variety of applications in protein engineering and drug discovery. A new thematic miniseries on intein-mediated protein splicing appeared in a recent issue of The Journal of Biological Chemistry.
In the first minireview, Olga Novikova, Natalya Topilina and Marlene Belfort discuss the overall function of inteins and the sporadic distribution of inteins among closely related species. Overall, there is a bias for inteins to insert into proteins involved in DNA metabolism, such as polymerases, helicases and topoisomerases. In addition, inteins normally are located at protein active sites or in key ligand-binding surfaces. While the rationale for intein localization is still a matter of debate, the authors mention the importance of intein retention. Improper removal of an intein within a conserved protein motif would be deleterious and therefore ensures retention of an active intein for viability of the host. Understanding the evolution and distribution of inteins will shed light on the possibility that inteins function as unique regulatory elements.
In the second minireview, Kenneth V. Mills, Margaret A. Johnson and Francine B. Perler focus on the wide variety of splicing mechanisms. Inteins have evolved to regulate tightly the steps of splicing; however, inteins don’t use a universal mechanism. This review discusses the general strategies for catalysis and the roles of various amino acids during splicing. While the basic steps in protein splicing have been known since the 1990s, how the reactions are coordinated is still unknown. Detailed kinetic and structural studies are needed of multiple inteins to determine whether catalytic strategies are universal or specific to a subset of inteins.
Ertan Eryilmaz, Neel Shah, Tom Muir and David Cowburn explore in the third minireview the structural features of inteins and the variability in splicing mechanisms. While all inteins share the same fold and have highly conserved sequence motifs, inteins have surprisingly different splicing efficiencies. This review describes the structural basis of protein splicing, intein dependence on exteins for protein splicing and distal mutations that affect protein splicing. Allosteric networks, the authors conclude, may play a larger role in determining intein activity than previously thought, because mutations distal from the active site can modulate intein activity.
In the final minireview, David W. Wood and Julio A. Camarero share advances in the applications of inteins. Early engineered inteins were used in protein purification, but now optimized trans-splicing and trans-cleaving inteins have enabled a wide variety of applications in protein labeling, metabolic engineering, biomaterials construction, intein-based biosensors, gene delivery and protein cyclization. These new techniques allow specific control over biological functions of proteins in living cells, plants and whole animals. Future applications will build on these techniques and promise to reveal new classes of therapeutic proteins. It is exciting to see movement out of the laboratory and into real-world applications in the fields of energy and medicine, among others.
The four minireviews in this series help to broaden our thinking about protein splicing. In an editorial commentary, Perler and Norma M. Allewell conclude that significant progress has been made to better understand intein mechanisms; however, there are still many unanswered questions. While evolutionary biologists question whether inteins are selfish elements and biochemists seek to understand how inteins work, intein-mediated protein splicing creates opportunities in many scientific areas. The numerous intein applications have huge potential for modifying, synthesizing and controlling protein function in the future.

Jenna HendershotJenna Hendershot (hendeje@
umich.edu) earned a B.S. in cellular and molecular biology from Grand Valley State University and is completing her Ph.D. in biological chemistry at the University of Michigan.