Research

Matthew Gage Research

Structure/Function Relationships in Titin

Titin, the largest known protein, plays an integral role in sarcomere structure, both as a scaffold and as a regulator of both passive and active force.  Titin is composed of a series of repeated immunoglobulin and fibronectin domains with a large disordered region in the center of the protein (Figure 1).  Our lab has been working to understand three primary principles involving the relationship between titin structure and it’s function.

  1. How does the structure of the N2A region dictate the function of this region of titin?
  2. What is the relationship between sequence of the PEVK region and the function of this region in both active and passive force?
  3. What are the control mechanisms associated with titin splicing and what are the evolutionary relationships between individual domains in titin?
Structure/Function Relationships involving the N2A Region of titin
The extensible region of titin is the I-band region, between the Z-disk and the thick filament. This region is divided into three segments. The first region consists of a series of repeated immunoglobulin domains that become separated under low force eccentric stretching.  The second region is a disordered region called the PEVK region due to its high proline, glutamic acid, valine and lysine content.  The final region is another region of repeated immunoglobulin domains.  Separating the initial immunoglobulin domain region and the PEVK region is a region called the N2A region, which consists of four immunoglobulin domains with a unique sequence region between the first and second immunoglobulin domain. This region has been implicated in a number of different interactions with other proteins and has been the focus of study in our lab. The majority of domains in titin have a beta-sheet structure but work in our lab has shown that the insertion sequence in the N2A region has an alpha-helical core with significant unordered structure around it. Current work is focused on trying to elucidate a high resolution structure of this region. Similar studies are underway with the three immunoglobulin domains between the insertion sequence and the PEVK region. We have also been investigating the stability of the individual domains to understand how domain stability can contribute to the mechanical force generated by titin. The role of titin in active force has begun to be more widely accepted and recent work involving our lab has demonstrated that the N2A region is capable of binding to actin filaments. This interaction becomes stronger in the presence of calcium, suggesting that it may be important for titin’s role in active muscle. Current work is focused on deciphering which domains in the N2A region are critical for this interaction.

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Conformation and Passive Force
          The PEVK region is located in the middle of the I-band sequence of titin and plays a critical role during muscle fiber elongation.  The PEVK region is composed of two sequence motifs, called the PPAK and poly-E motifs.  The PPAK motif is a 28 amino acid sequence that is proline and lysine rich.  This motif is identified by the PPAK sequence that is at the beginning of the motif. The poly-E motif is glutamate rich with a clusters of 2-4 glutamates throughout the sequence.  This region is encoded for by >100 exons, which are spliced together differently in different types of muscles, making the PEVK region the region of highest alternative spicing in titin.          One interesting feature of this region is that it does not contain a regular, stable structure due to the high charge density and low hydrophobicity. Due to this property, this region is classified as an Intrinsically Disordered Region (IDR). While this region does not have an ordered structure, it it capable of generating high force per distance stretched. Current models suggest that this force is developed through both enthalpic and entropic contributions from the two sequence motifs. Work in our lab has been focused on understanding the relationship between sequence variations of the two motifs and function and the impact of the surrounding solution on conformation. We are starting to explore more deeply the relationship between sequence and mechanical force and the mechanisms controlling splicing of this region with the goal of understanding how the sequence of this region impacts the function of titin in different types of muscles.

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Bioinformatics and Evolutional Studies on Titin
The bioinformatics revolution has provided an exciting toolbox of approaches to use big data analysis to explore the relationship between gene expression, protein levels and physiological conditions.  Our lab has been using RNA-Seq and Tag-Seq approaches to determine differential gene expression patterns in muscle cells.  Questions that we are exploring include the effect of different nutritional sources on muscle gene expression, the differences in gene expression in the mdm mouse model for muscular dystrophy and expression differences due to damage associated with eccentric stretch. We are also using these data sets to identify differential splicing patterns and to understand the splicing control mechanisms. The size of titin and the diversity of its domains raises interesting questions regarding the evolutionary relationships between the various repeated domains. One particular point of interest are the regions of titin that exhibit evolutionary repeats.  These are regions where a series of repeated domains do not show evolutionary relationships to each other but this unit is repeated several times and each position in the repeat is evolutionarily related to the domain in the same position in each repeat.  We are exploring how these repeated regions evolved and whether there is a functional significance to them.

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Antimicrobial Development

The growing rate of antimicrobial resistance has become a major health issue worldwide. A recent study by the RAND institute predicts that, if left unchecked, the world population will be 11 to 444 million people smaller in 2050 than if there was no antimicrobial resistance. This decreased population would reduce the overall global economy by up to 3% or ~$125 trillion. Currently, the cost associated with treating antibiotic resistant infections is estimated at $2 billion annually, which has more than doubled in the past 15 years, and these costs will continue to rise in the future, further impacting the U.S. and global economy if they remain unchecked.

            There are two issues that are major roadblocks to addressing the growing rate of resistance. First, there are a limited number of new antimicrobial compounds that have either been approved or that are in the development pipeline (51 compounds are currently in the pipeline). The return on investment is limited because antibiotics are only prescribed for a short period of time compared to more chronic diseases such as cancer or HIV, where patients might take a drug for years. This has been a deterrent for major pharmaceutical companies to invest in development of new compounds. Second, development has focused on a limited number of targets, which allows resistance to rapidly develop following introduction of a new antibiotic into therapeutic use.

            Work in our lab is focused on identifying new targets for drug development studies. The current area of focus in on a novel transcription factor conserved within the bacterial kingdom.  Our lab has developed fluorescence-based reporter assay to monitor transcription factor activity and we have used this assay to screen potential inhibitors.  Our current work is focused on characterizing the binding of these potential inhibitors to our target transcription factor using a variety of biophysical techniques.

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