Uncovering One-Dimensional Reaction Coordinate that Underlies Structure-Function Relationship of Proteins

Understanding the mechanism of functional protein dynamics is critical to understanding protein functions. Reaction coordinates is a central topic in protein dynamics and the grail is to find the one-dimensional reaction coordinate that can fully determine the value of committor (i.e. the reaction probability in configuration space) for any protein configuration. We present a powerful new method that can, for the first time, identify the rigorous one-dimensional reaction coordinate in complex molecules. This one-dimensional reaction coordinate is determined by a fundamental mechanical operator--the generalized work functional. This method only requires modest computational cost and can be readily applied to large molecules. Most importantly, the generalized work functional is the physical origin of the collectivity in functional protein dynamics and provides a tentative roadmap that connects the structure of a protein to its function.

Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans

Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.

Resonant fluctuations of selection pressure exponentially accelerate fitness valley crossing

Two functional protein sequences can sometimes be separated by a fitness valley - a series of low or non-functional intermediate mutations that must be traversed to reach a more optimal or refined function. Time-varying selection pressure modulates evolutionary sampling of such valleys. Yet, how the amplitude and frequency of fluctuating selection influence the rate of protein evolution is poorly understood. Here, we derive a simple equation for the time-dependent probability of crossing a fitness valley as a function of evolutionary parameters: valley width, protein size, mutation rate, and selection pressure. The equation predicts that, under low selection pressure, the valley crossing rate is magnified by a factor that depends exponentially on valley width. However, after a characteristic time set by the evolutionary parameters, the rate rapidly decays. Thus, there is an optimal frequency of selection-pressure fluctuations that maximizes the rate of protein optimization. This result is reminiscent of the resonance frequency in mechanical systems. The equation unites empirical and theoretical results that were previously disconnected, and is consistent with time-dependent in vitro and clinical data. More generally, these results suggest that seasonal and climate oscillations could synchronously drive protein evolution at the resonant frequency across a range of organism hosts and timescales. This theory could also be applied to optimize de novo protein evolution in laboratory directed evolution using time-varying protocols.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

Genome-Wide Identification and Characterization of the Trehalose-6-phosphate Synthetase (TPS) Gene Family in Watermelon (Citrullus lanatus) and Their Transcriptional Responses to Salt Stress

With the increase in watermelon cultivation area, there is an urgent need to explore enzymatic and genetic resources for the sustainable development of watermelon, especially under salt stress. Among the various compounds known, trehalose plays an important role in regulating abiotic stress tolerances in diverse organisms, including plants. Therefore, the present study comprehensively analyzed the trehalose-6-phosphate synthase (TPS) gene family in watermelon. The study analyzed the functional classification, evolutionary characteristics, and expression patterns of the watermelon TPS genes family. Seven ClTPSs were identified and classified into two distinct classes according to gene structure and phylogeny. Evolutionary analysis suggested the role of purifying selection in the evolution of the TPS family members. Further, cis-acting elements related to plant hormones and abiotic stress were identified in the promoter region of the TPS genes. The tissue-specific expression analysis showed that ClTPS genes were widely expressed in roots, stems, leaves, flowers, and fruits, while ClTPS3 was significantly induced under salt stress. The overexpression of ClTPS3 in Arabidopsis thaliana significantly improved salt tolerance. Finally, the STRING functional protein association networks suggested that the transcription factor ClMYB and ClbHLH regulate ClTPS3. Thus, the study indicates the critical role of ClTPS3 in watermelon response to salt stress.

XAB2 Dynamics during DNA Damage-Dependent Transcription Inhibition

Xeroderma Pigmentosum group A (XPA)-binding protein 2 (XAB2) is a multi-functional protein that plays a critical role in distinct cellular processes including transcription, splicing, DNA repair and mRNA export. In this study, we detailed XAB2 involvement during Nucleotide Excision Repair (NER), a repair pathway that guarantees genome integrity against UV light-induced DNA damage and that specifically removes transcription-blocking damage in a dedicated process known as Transcription-Coupled repair (TC-NER). Here, we demonstrated that XAB2 is involved specifically and exclusively in TC-NER reaction and solely for RNA Polymerase 2 transcribed genes. Surprisingly, contrary to all the other NER proteins studied so far, XAB2 does not accumulate on the local UV-C damage but on the contrary is remobilized after damage induction. This fast change in mobility is restored when DNA repair reactions are completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have identified that XAB2 is detached after DNA damage induction from the DNA:RNA hybrids, commonly known as R-loops, and the CSA and XPG protein and this release is thought to contribute to the DNA damage recognition step during TC-NER. Importantly, we have disclosed a role for XAB2 in retaining RNAP2 on its substrate.

A Glance into MTHFR Deficiency at a Molecular Level

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.