Targeting Biomolecular Condensation and Protein Aggregation against Cancer.

Biomolecular condensates, membrane-less entities arising from liquid-liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.

Water Leakage Pathway Leads to Internal Hydration of the p53 Core Domain.

The gene encoding the p53 tumor suppressor protein is the most frequently mutated oncogene in cancer patients; yet, generalized strategies for rescuing the function of different p53 mutants remain elusive. This work investigates factors that may contribute to the low inherent stability of the wild-type p53 core domain (p53C) and structurally compromised Y220C mutant. Pressure-induced unfolding of p53C was compared to p63C, the p53 family member with the highest stability, the engineered superstable p53C hexamutant (p53C HM), and lower stability p53C Y220C cancer-associated mutant. The following pressure unfolding values (P50% bar) were obtained: p53C 3346, p53C Y220C 2217, p53C HM 3943, and p63C 4326. Molecular dynamics (MD) simulations revealed that p53C Y220C was most prone to water infiltration, followed by p53C, whereas the interiors of p53C HM and p63C remained comparably dry. A strong correlation (r2 = 0.92) between P50% and extent of interior hydration was observed. The pathways of individual water molecule entry and exit were mapped and analyzed, revealing a common route preserved across the p53 family involving a previously reported pocket, along with a novel surface cleft, both of which appear to be targetable by small molecules. Potential determinants of propensity to water incursion were assessed, including backbone hydrogen bond protection and combined sequence and structure similarity. Collectively, our results indicate that p53C has an intrinsic susceptibility to water leakage, which is exacerbated in a structural class mutant, suggesting that there may be a common avenue for rescuing p53 function.

Conformational stability of SARS-CoV-2 glycoprotein spike variants.

The severe acute respiratory syndrome spread worldwide, causing a pandemic. SARS-CoV-2 mutations have arisen in the spike, a glycoprotein at the viral envelope and an antigenic candidate for vaccines against COVID-19. Here, we present comparative data of the glycosylated full-length ancestral and D614G spike together with three other transmissible strains classified by the World Health Organization as variants of concern: beta, gamma, and delta. By showing that D614G has less hydrophobic surface exposure and trimer persistence, we place D614G with features that support a model of temporary fitness advantage for virus spillover. Furthermore, during the SARS-CoV-2 adaptation, the spike accumulates alterations leading to less structural stability for some variants. The decreased trimer stability of the ancestral and gamma and the presence of D614G uncoupled conformations mean higher ACE-2 affinities compared to the beta and delta strains. Mapping the energetics and flexibility of variants is necessary to improve vaccine development.

The chameleonic behavior of p53 in health and disease: the transition from a client to an aberrant condensate scaffold in cancer.

In 1972, the Weber statement, "The multiplicity of interactions and the variety of effects that follow from them show that multimer proteins are unlikely to be limited to a minimal number of allowed conformations," first addressed the dynamic nature of proteins. This idea serves as a foundation for understanding why several macromolecules, such as p53, exhibit the properties of a molecular chameleon. Functionally competent states comprise a myriad of p53 three-dimensional arrangements depending on the stimuli. For instance, the interaction of p53 with nuclear components could induce liquid-liquid phase separation (LLPS) and the formation of membraneless organelles. The functional or deleterious role of p53 in liquid droplets is still unclear. Functional aspects display p53 interconverting between droplets and tetramer with its functional abilities maintained. In contrast, the aberrant phase separation is likely to fuel the aggregation path, usually associated with the onset and progression of age-related neurodegenerative diseases and cancer. Here, we gathered the most relevant aspects that lead p53 to phase separation and the resulting structural effects, attempting to understand p53's functional and disease-relevant processes. Aberrant phase separation and aggregation of mutant p53 have become important therapeutic targets against cancer.

Evolutionary Role of Water-Accessible Cavities in Src Homology 2 (SH2) Domains.

Protein excited states are fundamental in the understanding of biological function, despite the fact they are hardly observed using traditional biophysical methodologies. Pressure perturbation coupled with nuclear magnetic resonance (NMR) spectroscopy is a powerful physicochemical tool to glance at these low-populated high-energy states on a residue-by-residue basis and underpin mechanistic insights into protein functionalities. Here we performed pressure titrations using NMR spectroscopy and relaxation dispersion experiments to identify the low-lying energetic states of the c-Abl SH2 domain. By showing that the SH2 excited state contains a hydrated hydrophobic cavity, fast-exchange motions, and highly conserved residues facing the water-accessible hole, we discuss the implications of water-protein interactions in SH2 modules achieving high-affinity binding and promiscuous phospho-Tyr peptide recognition.

Protein of a thousand faces: The tumor-suppressive and oncogenic responses of p53.

The p53 protein is a pleiotropic regulator working as a tumor suppressor and as an oncogene. Depending on the cellular insult and the mutational status, p53 may trigger opposing activities such as cell death or survival, senescence and cell cycle arrest or proliferative signals, antioxidant or prooxidant activation, glycolysis, or oxidative phosphorylation, among others. By augmenting or repressing specific target genes or directly interacting with cellular partners, p53 accomplishes a particular set of activities. The mechanism in which p53 is activated depends on increased stability through post-translational modifications (PTMs) and the formation of higher-order structures (HOS). The intricate cell death and metabolic p53 response are reviewed in light of gaining stability via PTM and HOS formation in health and disease.

Nucleic acid actions on abnormal protein aggregation, phase transitions and phase separation.

Liquid-liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.

Polyclonal F(ab')2 fragments of equine antibodies raised against the spike protein neutralize SARS-CoV-2 variants with high potency.

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:106, and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT90). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab')2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab')2 to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab')2 was similar against the WT and P.2 variants, but displaced to higher concentrations by 0.39 log units against the P.1 (Gamma) variant. These results support the possibility of using equine F(ab')2 preparation for the clinical treatment of COVID patients.

Process intensification for the production of yellow fever virus-like particles as potential recombinant vaccine antigen.

Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy.

Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca2+-regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix (TNNC1-p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca2+ binding. We demonstrate cTnC's αD-helix normally functions as a central hub that controls minimally frustrated interactions, maintaining evolutionarily conserved rigidity of the N-domain. αD-helix perturbation remotely alters conformational dynamics of the N-domain, compromising its structural rigidity. Transgenic mice carrying this cTnC mutation exhibit altered dynamics of sarcomere function and hypertrophic cardiomyopathy. Together, our data suggest that disruption of evolutionary conserved molecular frustration networks by a myofilament protein mutation may ultimately compromise contractile performance and trigger hypertrophic cardiomyopathy.

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.

Exploring the polymorphism, conformational dynamics and function of amyloidogenic peptides and proteins by temperature and pressure modulation.

Our understanding of amyloid structures and the mechanisms by which disease-associated peptides and proteins self-assemble into these fibrillar aggregates, has advanced considerably in recent years. It is also established that amyloid fibrils are generally polymorphic. The molecular structures of the aggregation intermediates and the causes of molecular and structural polymorphism are less understood, however. Such information is mandatory to explain the pathological diversity of amyloid diseases. What is also clear is that not only protein mutations, but also the physiological milieu, i.e. pH, cosolutes, crowding and surface interactions, have an impact on fibril formation. In this minireview, we focus on the effect of the less explored physical parameters temperature and pressure on the fibrillization propensity of proteins and how these variables can be used to reveal additional mechanistic information about intermediate states of fibril formation and molecular and structural polymorphism. Generally, amyloids are very stable and can resist harsh environmental conditions, such as extreme pH, high temperature and high pressure, and can hence serve as valuable functional amyloid. As an example, we discuss the effect of temperature and pressure on the catalytic activity of peptide amyloid fibrils that exhibit enzymatic activity.

The Status of p53 Oligomeric and Aggregation States in Cancer.

Despite being referred to as the guardian of the genome, when impacted by mutations, p53 can lose its protective functions and become a renegade. The malignant transformation of p53 occurs on multiple levels, such as altered DNA binding properties, acquisition of novel cellular partners, or associating into different oligomeric states. The consequences of these transformations can be catastrophic. Ongoing studies have implicated different oligomeric p53 species as having a central role in cancer biology; however, the correlation between p53 oligomerization status and oncogenic activities in cancer progression remains an open conundrum. In this review, we summarize the roles of different p53 oligomeric states in cancer and discuss potential research directions for overcoming aberrant p53 function associated with them. We address how misfolding and prion-like amyloid aggregation of p53 seem to play a crucial role in cancer development. The misfolded and aggregated states of mutant p53 are prospective targets for the development of novel therapeutic strategies against tumoral diseases.

Oncogenic Gain of Function in Glioblastoma Is Linked to Mutant p53 Amyloid Oligomers.

Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer.

Biophysical characterization of p53 core domain aggregates.

Aggregation is the cause of numerous protein conformation diseases. A common facet of these maladies is the transition of a protein from its functional native state into higher order forms, such as oligomers and amyloid fibrils. p53 is an essential tumor suppressor that is prone to such conformational transitions, resulting in its compromised ability to avert cancer. This work explores the biophysical properties of early-, mid-, and late-stage p53 core domain (p53C) aggregates. Atomistic and coarse-grained molecular dynamics (MD) simulations suggest that early- and mid-stage p53C aggregates have a polymorphic topology of antiparallel and parallel ß-sheets that localize to the core amyloidogenic sequence. Both topologies involve similar extents of interstrand mainchain hydrogen bonding, while sidechain interactions could play a role in regulating strand orientation. The free energy difference between the antiparallel and parallel states was within statistical uncertainty. Negative stain electron microscopy of mature fibrils shows a wide distribution of fiber widths, indicating that polymorphism may extend to the quaternary structure level. Circular dichroism of the fibrils was indicative of ß-sheet rich structures in atypical conformations. The Raman spectrum of aggregated p53C was consistent with a mixture of arranged ß-sheets and heterogeneous structural elements, which is compatible with the MD findings of an ordered ß-sheet nucleus flanked by disordered structure. Structural polymorphism is a common property of amyloids; however, because certain polymorphs of the same protein can be more harmful than others, going forward it will be pertinent to establish correlations between p53C aggregate structure and pathology.

The intrinsically disordered C terminus of troponin T binds to troponin C to modulate myocardial force generation.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

A packing for A-form DNA in an icosahedral virus.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

Alpha-synuclein stepwise aggregation reveals features of an early onset mutation in Parkinson's disease.

Amyloid formation is a process involving interconverting protein species and results in toxic oligomers and fibrils. Aggregated alpha-synuclein (αS) participates in neurodegenerative maladies, but a closer understanding of the early αS polymerization stages and polymorphism of heritable αS variants is sparse still. Here, we distinguished αS oligomer and protofibril interconversions in Thioflavin T polymerization reactions. The results support a hypothesis reconciling the nucleation-polymerization and nucleation-conversion-polymerization models to explain the dissimilar behaviors of wild-type and the A53T mutant. Cryo-electron microscopy with a direct detector shows the polymorphic nature of αS fibrils formed by heritable A30P, E46K, and A53T point mutations. By showing that A53T rapidly nucleates competent species, continuously elongates fibrils in the presence of increasing amounts of seeds, and overcomes wild-type surface requirements for growth, our findings place A53T with features that may explain the early onset of familial Parkinson's disease cases bearing this mutation.

Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus.

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main ß-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.

New Heteroleptic Ruthenium(II) Complexes with Sulfamethoxypyridazine and Diimines as Potential Antitumor Agents.

Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy)2smp](PF6) (1) and [Ru(phen)2smp](PF6) (2), in which smp = sulfamethoxypyridazine; bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1) or two phen (in 2) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine-one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes' preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.