Protein folding, misfolding, and un/non-folding: overview of the SP16 Session at the 20^th IUPAB congress, 45^th Annual Meeting of SBBf, and 50^th Annual Meeting of SBBq

Abstract

The classical and idealistic consideration of the protein universe as a set of well-ordered proteinaceous machines with unique spatial organizations conducting unique biological functions is changed because of the recognition that proteins can fold, misfold, or be disordered to different degree. Functional repertoires of intrinsically disordered proteins complement functions of ordered proteins, whereas protein misfolding and concomitant oligomerization, aggregation, and fibrillation are related to the pathogenesis of numerous human diseases. Flexible proteins can undergo liquid–liquid phase separation, which is at the heart of the biogenesis of numerous membrane-less organelles. Many of these aspects were highlighted in four talks delivered at the SP16 Session at the 20th IUPAB congress, 45th Annual Meeting of SBBf, and 50th Annual Meeting of SBBq.

Over a couple of last decades, our perception of a functional protein as a crystal-like machine with an active site in a form of a unique and rigid key-hole, which is evolutionary mastered to exactly fit the unique and rigid key (substrate) has changed. It is recognized now that protein functionality goes far beyond the classic “lock-and-key” and “induced fit” models, and a key biological idea that structure defines function is converted into the structure–function continuum model (Uversky 2016a, 2016b, 2019). This is mostly due to the recognition of the existence of intrinsically disordered proteins (IDPs) combined with the understanding that proteins can be functionally active not only in a well-folded globular state, but also in a partially or fully unfolded state (Dunker et al. 2001; Uversky et al. 2000; Wright and Dyson 1999; Tompa 2002). Similarly, our perception of the protein misfolding and aggregation as an annoying artifact complicating structural and functional analysis of proteins has changed as well, since these two events are at the heart of many pathological conditions (Dobson 2003; Uversky 2003). The interconnection between protein folding, misfolding, and non-folding is illustrated by Fig. 1 showing several examples of IDPs with different levels of disorder as representatives of the intrinsically disordered part of the protein universe, a well-folded protein machine (chaperone GroEL), and amyloid fibrils resulting from the assembly of folded and misfolded species, respectively. Finally, our understanding of the cellular compartmentalization as the existence of classical membrane-embedded organelles has changed to a new view, where liquid–liquid phase separation, often driven by the weak polyvalent interactions of IDPs with their partners, defines the biogenesis of numerous membrane-less organelles (Nesterov and Ilyinsky, 2021; Brangwynne et al. 2009; Hyman et al. 2014). All these aspects of modern protein science were touched upon at the SP16 Session at the 20^th IUPAB congress, 45^th Annual Meeting of SBBf, and 50^th Annual Meeting of SBBq.

Fig. 1.

Interconnectivity of protein folding, misfolding, and non-folding processes

Prof. Prakash Kulkarni from the City of Hope National Medical Center (Duarte, CA, USA) discussed interconnection between protein conformational dynamics and phenotypic switching (Kulkarni 2020; Kulkarni and Kulkarni 2019; Lin et al. 2019; Mooney et al. 2016; Kulkarni and Getzenberg 2016; Rangarajan et al. 2015). He pointed out that due to their capability to be engaged in promiscuous binding with multiple partners, IDPs often occupy hub positions in the scale-free cellular protein interaction networks (PINs). The highly dynamic nature of these proteins and their propensity for post-translational modifications contribute to the ‘conformational’ noise, where means leading to the increase in this conformational noise may trigger an increase in stochastic, ‘promiscuous’ interactions of IDPs, leading to the activation of latent pathways and can induce ‘rewiring’ of the PIN to yield an optimal output. These considerations emphasize the critical role of IDPs in regulating information flow and show how IDP conformational dynamics and conformational noise might facilitate cellular decision making.

Prof. Gonzalo de Prat-Gay from Fundación Instituto Leloir, IIB-BA Conicet (Buenos Aires, Argentina) dedicated his talk to the discussion of the role of the viral intrinsically disordered proteins, a phosphoprotein, P, and a nucleoprotein N, from respiratory syncytial virus (RSV) in liquid–liquid phase separation (LLPS) leading to the formation of “viral factories”, which are liquid-like structures within the cytosol of infected cells and sites for transcription and replication (Lopez et al. 2021). He showed that purified P undergoes homotypic LLPS, where the C-terminal molten globule-like (MG) domain plays an important role. Furthermore, mixing P and N triggers heterotypic LLPS at remarkably low concentrations of these proteins. When co-transfect into a cell, P and N yield small liquid granules that can coalesce to yield large granules within the cell. The time course of infection experiments revealed that small granular nuclei can grow in size to render large viral factory granules observed for RSV and other mononegavirales. It is concluded that the minimal set of components sufficient for the biological LLPS leading to the formation of viral factory granules includes the N and P proteins.

Prof. Orkide Coskuner-Weber from Turkish-German University (Istanbul, Turkey) represented a computational biophysicist perspective of the two well-known conformational diseases, the Alzheimer’s and Parkinson’s diseases (AD and PD) (Fatafta et al. 2021; Akbayrak et al. 2020; Coskuner-Weber and Uversky 2018). Major players in these devastating maladies are IDPs — amyloid-β (Aβ) and α-synuclein, respectively, aggregation of which produce a broad spectrum of species ranging from dimers to fibrils. It was pointed out that the typical in vitro experimental analysis of pathological IDPs is conducted in rather artificial and idealistic conditions which are far from physiological, where the aggregation of pathological IDPs is affected by numerous biochemical reactions. Furthermore, such experimental analyses under the complex physiologic-like conditions should be complemented by computational studies. Next, recent advances in the research on Aβ and α-synuclein from a physico-chemical perspective, focusing on the physiological factors that influence aggregation processes of these proteins in AD and PD were discussed. The presentation was concluded by a detailed emphasis on the computational biophysics studies that provide grounds for better understanding of various in vivo factors on aggregation of Aβ and α-synuclein.

Finally, Dr. Alexander V. Fonin from the Institute of Cytology of the Russian Academy of Sciences (Saint Petersburg, Russia) represented a new view of the formation of PML (promyelocytic leukaemia) bodies (Fonin et al. 2021). Due to their exceptional biological significance for many cellular processes in health and disease, PML bodies are among the best-studied membrane-less organelles. It was long assumed that the process of PML body biogenesis is triggered by the oxidative dimerization of PML, where the formation of disulfide bonds between monomers initiates the formation of an insoluble “aggregate” to which client proteins are recruited via SUMO/SIM interactions. This view of PML-bodies originated long before the emergence of the fundamentally new ideas on the roles of liquid–liquid phase separation in the formation of membrane-less organelles. Proposed in this presentation a new view of the PML body biogenesis suggests that the formation of PML bodies is initiated as a result of liquid–liquid phase separation of PML isoforms, leading to the formation of liquid droplets, which appears due to the multiple weak nonspecific interactions of intrinsically disordered regions of PML isoforms. Next, insoluble scaffold is formed, to which, due to polyvalent, primarily SUMO/SIM interactions, client proteins are attracted, thereby forming a dynamic layer that exchanges its content with the environment.

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