Sequence Divergence and Functional Specializations of the Ancient Spliceosomal SF3b: Implications in Flexibility and Adaptations of the Multi-Protein Complex

Multi-protein assemblies are complex molecular systems that perform highly sophisticated biochemical functions in an orchestrated manner. They are subject to changes that are governed by the evolution of individual components. We performed a comparative analysis of the ancient and functionally conserved spliceosomal SF3b complex, to recognize molecular signatures that contribute to sequence divergence and functional specializations. For this, we recognized homologous sequences of individual SF3b proteins distributed across 10 supergroups of eukaryotes and identified all seven protein components of the complex in 578 eukaryotic species. Using sequence and structural analysis, we establish that proteins occurring on the surface of the SF3b complex harbor more sequence variation than the proteins that lie in the core. Further, we show through protein interface conservation patterns that the extent of conservation varies considerably between interacting partners. When we analyze phylogenetic distributions of individual components of the complex, we find that protein partners that are known to form independent subcomplexes are observed to share similar profiles, reaffirming the link between differential conservation of interface regions and their inter-dependence. When we extend our analysis to individual protein components of the complex, we find taxa-specific variability in molecular signatures of the proteins. These trends are discussed in the context of proline-rich motifs of SF3b4, functional and drug binding sites of SF3b1. Further, we report key protein-protein interactions between SF3b1 and SF3b6 whose presence is observed to be lineage-specific across eukaryotes. Together, our studies show the association of protein location within the complex and subcomplex formation patterns with the sequence conservation of SF3b proteins. In addition, our study underscores evolutionarily flexible elements that appear to confer adaptive features in individual components of the multi-protein SF3b complexes and may contribute to its functional adaptability.

RETINOBLASTOMA RELATED (RBR) interaction with key factors of the RNA-directed DNA methylation (RdDM) pathway

● Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs are recruited by AGO4 and serve as guides to direct AGO4-siRNA complexes to chromatin bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. In silico exploration of Arabidopsis RBR protein partners revealed that several members of the RdDM pathway contain a motif that confers high affinity binding to RBR, including the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2 and SUVR2. We demonstrate that RBR binds to DRM2, DRD1 and SUVR2. We also report that seedlings from loss-of-function mutants in RdDM and in RBR show similar phenotypes in the root apical meristem. Furthermore, we show that RdDM and SUVR2 targets are up-regulated in the 35S::AmiGO-RBR background. Our results suggest a novel mechanism for RBR function in transcriptional gene silencing based on the interaction with key players of the RdDM pathway and opens several new hypotheses, including the convergence of RBR-DRM2 on the transcriptional control of TEs and several cell/tissue and stage-specific target genes.

ALS2-Related Motor Neuron Diseases: From Symptoms to Molecules

Infantile-onset Ascending Hereditary Spastic Paralysis, Juvenile Primary Lateral Sclerosis and Juvenile Amyotrophic Lateral Sclerosis are all motor neuron diseases related to mutations on the ALS2 gene, encoding for a 1657 amino acids protein named Alsin. This ~185 kDa multi-domain protein is ubiquitously expressed in various human tissues, mostly in the brain and the spinal cord. Several investigations have indicated how mutations within Alsin’s structured domains may be responsible for the alteration of Alsin’s native oligomerization state or Alsin’s propensity to interact with protein partners. In this review paper, we propose a description of differences and similarities characterizing the above-mentioned ALS2-related rare neurodegenerative disorders, pointing attention to the effects of ALS2 mutation from molecule to organ and at the system level. Known cases were collected through a literature review and rationalized to deeply elucidate the neurodegenerative clinical outcomes as consequences of ALS2 mutations.

Novel Hemoglobin from Synechocystis sp. PCC 6803: Shedding Light on the Structure-Function Relationship and Its Biotechnological Applications

Cyanobacteria are oxygenic photosynthetic prokaryotes, practically present in every plausible environment on the earth. In 1996, the first cyanobacterial genome was sequenced from Synechocystis sp. PCC 6803 and the cyanobacterial genome database has been continuously growing with genomes from more than 300 cyanobacterial and other related species, so far. Synechocystis sp. PCC 6803 is one of the best-characterized cyanobacteria and has developed into a model cyanobacterium that scientists are using throughout the world. At the same time, the field of hemoglobin was undergoing a breakthrough with the identification of new globins in all three kingdoms of life including cyanobacteria. Since then, the newly identified globins in the cyanobacteria are raising intriguing questions about their structure and physiological functions, which are quite different from vertebrate’s hemoglobin and myoglobin. These hemoglobins have displayed unprecedented stability, unique heme coordination, novel conformational changes, and other properties that are not often observed in the globin superfamily. This chapter provides an overview of the unique globin from Synechocystis sp. PCC 6803, its interacting protein partners, proposed functions, and its biotechnological implications including potential in the field of artificial oxygen carriers.

Two Liberibacter Effectors Combine to Suppress Critical Innate Immune Defenses and Facilitate Huanglongbing Pathogenesis in Citrus

Genome sequence analyses predicted the presence of effectors in the gram-negative Candidatus Liberibacter asiaticus (CLas) even without the presence of a classical type III secretion system. Since CLas is not culturable, it is not possible to perform traditional gene knockout experiments to determine the role of various effectors in Huanglongbing (HLB) pathogenesis. Therefore, we followed an alternative functional genomics approach to examine the role of the CLas effectors in HLB pathogenesis in general and more specifically in suppressing citrus innate immune response. Here, we focused on the CLas effectors, P235 and Effector 3, to perform the following studies. First, proteomic studies by LC-MS/MS were conducted to screen the putative interacting citrus protein partners of P235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and the most probable candidates were identified based upon their high protein scores from LC-MS/MS. Second, a transgenic tobacco split GFP system was designed for in planta detection of the most probable citrus interacting protein partners of P235 and Effector 3. Third, in vitro and in planta studies were performed to show that each of two effectors interacts with and inhibits the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species (ROS), blocking of bactericidal lipid binding protein (LTP), and induction of premature programmed cell death (PCD), thereby supporting CLas infection and HLB pathogenesis. Finally, an LTP mimic was designed to sequester and block the CLas effector and to rescue the bactericidal activity of LTP.

Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

Correlations from structure and phylogeny combine constructively in the inference of protein partners from sequences

Inferring protein-protein interactions from sequences is an important task in computational biology. Recent methods based on Direct Coupling Analysis (DCA) or Mutual Information (MI) allow to find interaction partners among paralogs of two protein families. Does successful inference mainly rely on correlations from structural contacts or from phylogeny, or both? Do these two types of signal combine constructively or hinder each other? To address these questions, we generate and analyze synthetic data produced using a minimal model that allows us to control the amounts of structural constraints and phylogeny. We show that correlations from these two sources combine constructively to increase the performance of partner inference by DCA or MI. Furthermore, signal from phylogeny can rescue partner inference when signal from contacts becomes less informative, including in the realistic case where inter-protein contacts are restricted to a small subset of sites. We also demonstrate that DCA-inferred couplings between non-contact pairs of sites improve partner inference in the presence of strong phylogeny, while deteriorating it otherwise. Moreover, restricting to non-contact pairs of sites preserves inference performance in the presence of strong phylogeny. In a natural dataset, as well as in realistic synthetic data based on it, we find that non-contact pairs of sites contribute positively to partner inference performance, and that restricting to them preserves performance, evidencing an important role of phylogeny.

DLA-Ranker: Evaluating protein docking conformations with many locally oriented cubes.

Proteins ensure their biological functions by interacting with each other, and with other molecules. Determining the relative position and orientation of protein partners in a complex remains challenging. Here, we address the problem of ranking candidate complex conformations toward identifying near-native conformations. We propose a deep learning approach relying on a local representation of the protein interface with an explicit account of its geometry. We show that the method is able to recognise certain pattern distributions in specific locations of the interface. We compare and combine it with a physics-based scoring function and a statistical pair potential.

Functional Genomics of PRUNE1 in Neurodevelopmental Disorders (NDDs) Tied to Medulloblastoma (MB) and Other Tumors

We analyze the fundamental functions of Prune_1 in brain pathophysiology. We discuss the importance and maintenance of the function of Prune_1 and how its perturbation influences both brain pathological conditions, neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA; OMIM: 617481), and tumorigenesis of medulloblastoma (MB) with functional correlations to other tumors. A therapeutic view underlying recent discoveries identified small molecules and cell penetrating peptides to impair the interaction of Prune_1 with protein partners (e.g., Nm23-H1), thus further impairing intracellular and extracellular signaling (i.e., canonical Wnt and TGF-β pathways). Identifying the mechanism of action of Prune_1 as responsible for neurodevelopmental disorders (NDDs), we have recognized other genes which are found overexpressed in brain tumors (e.g., MB) with functional implications in neurodevelopmental processes, as mainly linked to changes in mitotic cell cycle processes. Thus, with Prune_1 being a significant target in NDDs, we discuss how its network of action can be dysregulated during brain development, thus generating cancer and metastatic dissemination.