Despite major advances in discerning protein structure and function, a large fraction of the ‘protein universe’ still remains elusive and is often referred to as the ‘dark proteome’. Intrinsically disordered proteins or IDPs are proteins that lack a rigid 3D structure, and are a major component of this dark proteome present in all three kingdoms of life. IDPs play critical roles in numerous biological processes and are implicated in the emergence of new traits and adaptive opportunities. Thus, a deeper understanding of these enigmatic molecules can not only help illuminate the dark side of the protein universe but may also uncover novel therapeutic targets for many chronic ailments.
1. What are IDPs and why are they so interesting?
A good portion of the ‘protein universe’ embodies the ‘dark proteome’, comprising of proteins not amenable to experimental structure determination by existing means (such as X-ray crystallography) and inaccessible to homology modeling. Hence, the dark proteome has remained largely unappreciated. Intrinsically disordered proteins (IDPs), proteins that lack rigid structure, are a major component of this dark proteome across all three kingdoms of life. In fact, per some estimates, ~50% of the proteins in our cells, are IDPs! Despite a lack of structure, IDPs play critical roles in numerous important biological processes such as gene transcription, splicing the heteronuclear RNA to produce mature messenger RNAs (mRNAs), intracellular signal transduction, cell cycle regulation, circadian rhythmicity, and even cell fate specification i.e., determining cellular phenotype, especially during development.
In fact, per some estimates, ~50% of the proteins in our cells, are IDPs!
Another interesting feature of IDPs is that while some become folded like regular proteins upon interacting with a partner protein or biomolecule, others can stochastically switch among distinct conformational states while remaining disordered. Finally, some IDPs can be functional even in a disordered state underscoring the extraordinary malleability of IDPs.
2. How big is the protein universe?
The distribution of matter and energy in the universe provides cosmologists with the principal source of information on its evolution, including its earliest stages. In a somewhat loose but perhaps appropriate analogy, structural biologists often speak of the ‘protein universe’, meaning the totality of all possible proteins. The total number of possible protein sequences is astronomical, and for all practical purposes, infinite! Every single protein arises from the combinatorial sequence of 20 amino acids, the building blocks. Thus, even if we assume an average protein length of 200 amino acids, there can be 20200 different protein sequences, a number that is much greater than the number of electrons in the entire universe!
Our current theoretical understanding of protein folding is insufficient to estimate the total possible number of protein structures, but it is likely to be vast. Consider this, assuming there are 10 million species on Earth and the genome of each species consists of 5,000 genes (an intermediate number between prokaryotes and eukaryotes), there are 5×1010 unique protein sequences. Besides, this estimate does not take into account the IDPs! Given that they exist as interchangeable ensembles, this number can be several orders of magnitude higher than 5×1010 and perhaps, could be closer to the number of stars in our universe – the observable universe contains an estimated 1022 to 1024 stars!
3. Why is malleability of the IDPs important and how does it contribute to stochasticity?
Since IDPs do not conform to a unique 3D structure with limited mobility but instead exist as ensembles of rapidly interconverting forms, they can interact with multiple partners unlike ordered proteins. Thus, malleability of IDPs is an important characteristic that they possess and is leveraged by the cell to rewire its protein interaction network (PIN). It is important to clarify that IDPs are naturally occurring proteins in our bodies and are integral components of the cell’s PIN. PINs are notformed by random interactions; on the contrary they are organized as scale-free networks in which the IDPs occupy critical ‘hub’ positions. Thus, the PIN is a defining feature of a given phenotype. Therefore, under physiological conditions, IDPs are tightly regulated. However, if their expression is deregulated and the IDPs are overexpressed, they can engage in ‘promiscuous’ interactions that can rewire the PIN to result in pathological states. Similarly, their under expression can also lead to PIN rewiring and altered physiological states (such as cancer, diabetes and neurodegenerative disease).
4. What are scale-free networks?
As demonstrated by Barabasi and colleagues as well as by several other researchers, power laws describe the distribution of various quantities in biological networks. These distributions have specific mathematical properties and hence, are described as scale-free networks, that is, networks in which the frequency distribution of node degrees (the number of nodes to which a given node is connected) follows a power law. The general pattern of network evolution that ensures scale-free behaviour is preferential attachment, where the probability of a node acquiring a new connection is proportional to the degree (the number of connections) of that node. Metaphorically, this can be described as a situation in which ‘the rich get richer’ or, from a selectionist perspective, ‘the fit get fitter’. A remarkable feature of scale-free networks is their resilience to perturbations.
5. What is biological noise and how do IDPs contribute to it?
It is now well established that gene expression is an intrinsically stochastic process, which often results in substantial “noise” in the system manifested as cell-to-cell variability in protein levels in a population. Thus, biological noise may be defined as random variability in quantities arising in biological systems even when they have identical genomes.
Our research has led to fundamentally new thinking – IDP conformational dynamics can give rise to ‘conformational noise’, which can further amplify transcriptional noise in the system since most, if not all transcription factors are IDPs. Thus, when deregulated, noise driven by IDPs can push a cell to switch its phenotype via a non-genetic mechanism of the rewiring of PIN, for example from a normal non-malignant phenotype to a malignant phenotype, or a drug sensitive to a drug-resistant phenotype. Given that ~80% of all cancer-associated proteins are IDPs, as are the factors necessary for reprogramming stem cells (the ‘Yamanaka factors’) in regenerative medicine, this new thinking can have significant impact. It not only sheds new light on the role of IDPs in the cancer cell’s bet-hedging strategies but also underscores the role of IDPs in non-genetic mechanisms underlying phenotypic heterogeneity inherent in cancers. Furthermore, non-genetic mechanisms driving phenotypic switching have broader implication both in biology and medicine.
6. Why were IDPs recently named amongst top 10 emerging technology?
In addition to their biological functions mentioned above, IDPs serve as crucial constituents of proteinaceous membrane-less organelles (PMLOs). PMLOs are formed by liquid-liquid phase separation when a polypeptide coalesces into a dense phase in an aqueous solution. These PMLOs are believed to have in vivo functional roles in cellular processes ranging from stress responses to regulation of gene expression and very likely in prebiotic evolution of the predecessor of first universal common ancestor.
A deeper understanding of IDPs can also shed new light on their role in phenotypic switching and the emergence of new traits and adaptive opportunities via non-genetic, protein-based mechanisms that have implications in biology and medicine, for example, in drug-resistance in cancer and the role of Prions in mad cow disease.
A deeper understanding of IDPs can also shed new light on their role in phenotypic switching, the emergence of new traits and adaptive opportunities via non-genetic, protein-based mechanisms
To create more awareness and motivate scientists to work on IDPs, we recently presented the Janus Challenge. We believe, meeting this challenge may not only shed new light and provide an alternative to the RNA world hypothesis (IDPs, rather than RNAs were the early biomolecules in the evolution of life), but it may also serve as an impetus for technological advances with important biomedical applications.
7. What is the ultimate goal of our research on IDPs?
Our long-term goals are to understand how a normal cell is transformed into a malignant one by factors from within as is seen in sporadic cancers (for example, prostate, lung, breast, pancreatic, and colon cancer), and how a cancer cell progresses to colonize distant locations, acquire stem cell-like properties, and develop drug resistance. More importantly, our goal is to tease out how ‘structural’ plasticity of the IDPs at the molecular level modulates phenotypic plasticity at the cellular level? Whether these phenotypic changes can be reversed? This knowledge can be used to gain a deeper understating of cancer, and develop new and effective cancer therapeutics.
Conclusion
Although IDPs are perceived as proteins that lack structure and therefore tacitly presumed to defy Anfinsen’s dogma – which postulates that protein structure defines function – it is important to note that IDPs are NOT random coils! It is true that IDPs lack secondary structure and exist as ensembles under physiological conditions at least in vitro. However, the ensembles do exhibit conformational preferences and therefore, IDPs DO HAVE ‘structure’ no matter how subtle. Thus, in contrast to prevailing wisdom, I believe Anfinsen’s paradigm still holds for IDPs. I think that perhaps structure, like beauty, lies in the eyes of the beholder.
____________________________________________________________________
Author:
Dr. Prakash Kulkarni is a Research Professor at the City of Hope National Medical Centre. After receiving his PhD in biochemistry from India, he completed his postdoctoral training in cell biology at New York University School of Medicine. He began his independent academic career as an Assistant Professor of urology and oncology at Johns Hopkins University School of Medicine. He then moved as an Associate Research Professor to the W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research. Prior to Johns Hopkins, Dr. Kulkarni held Staff Scientist positions in the Division of Chemistry & Chemical Engineering, and Division of Biology & Biological Engineering at the California Institute of Technology, and in the Department of Genetics at the Yale University School of Medicine. He is an Editorial Board Member of several Journals and also is an Assoc. Editor-in-Chief of Biomolecules. In addition, he is a member of Organizing Committees of International Meetings and serves as a scientific expert to various government and private organizations in the US, Europe, and Australia. His research interests are interdisciplinary across spatiotemporal scales and are focused on understanding how conformational dynamics of intrinsically disordered proteins contributes to phenotypic switching, especially in evolution of multicellularity, disease pathology and in non-genetic heterogeneity in cancer. Dr. Kulkarni is a Fellow of the Royal Society of Biology, UK.
Editors:
Sumbul Jawed Khan is a Ph. D. in Biological Sciences and Bioengineering from the Indian Institute of Technology Kanpur, where she studied the role of microenvironment in cancer progression and tumor formation. During her post-doctoral research at the University of Illinois at Urbana-Champaign, she investigated the gene regulatory networks that are important for tissue regeneration after damage or wounding. She is committed to science outreach and communication and believes it is essential to inspire young people to apply scientific methods to tackle the challenges faced by humanity. As an editor, her aim is to simplify, translate, and excite people about current advances in science.
Amrita Anand is in her 4th year of Ph.D. in Genetics and Genomics at the Baylor College of Medicine, Houston. She studies the reprogramming potential of certain key factors in the regeneration of mouse inner ear hair cells. She has been actively pursuing Science communication over the last three years as she enjoys bridging the gap between scientists and non-experts. As an editor, she wants to make science more accessible to the public and also hopes the hard work behind the science gets due credit.
Illustrator:
Duygu Koldere Vilain is a science illustrator who has a master’s degree in Molecular Biology and Genetics. While pursuing a Ph.D. she decided to change the path and switched into the science illustration area. Since then she is working with scientists to convey their messages in a clear and efficient way. She is happy to have merged her two passions: science and art. Follow her on Instagram https://www.instagram.com/dkv_scientific/
——————————————————————————————————
The contents of Club SciWri are the copyright of Ph.D. Career Support Group for STEM PhDs (A US Non-Profit 501(c)3, PhDCSG is an initiative of the alumni of the Indian Institute of Science, Bangalore. The primary aim of this group is to build a NETWORK among scientists, engineers, and entrepreneurs).
This work by Club SciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
