Cytoskeletal Protein 4.1R in Health and Diseases.

The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's physical properties, including mechanical stability and deformability, through its interaction with spectrin-actin. Recent research has uncovered additional roles of 4.1R beyond its function as a linker between the plasma membrane and the membrane skeleton. It has been found to play a crucial role in various biological processes, such as cell fate determination, cell cycle regulation, cell proliferation, and cell motility. Additionally, 4.1R has been implicated in cancer, with numerous studies demonstrating its potential as a diagnostic and prognostic biomarker for tumors. In this review, we provide an updated overview of the gene and protein structure of 4.1R, as well as its cellular functions in both physiological and pathological contexts.

Field and magic angle spinning frequency dependence of proton resonances in rotating solids.

Proton detection in solid state NMR is continuously developing and allows one to gain new insights in structural biology. Overall, this progress is a result of the synergy between hardware development, new NMR methodology and new isotope labeling strategies, to name a few factors. Even though current developments are rapid, it is worthwhile to summarize what can currently be achieved employing proton detection in biological solids. We illustrate this by analysing the signal-to-noise ratio (SNR) for spectra obtained for a microcrystalline α-spectrin SH3 domain protein sample by (i) employing different degrees of chemical dilution to replace protons by incorporating deuterons in different sites, by (ii) variation of the magic angle spinning (MAS) frequencies between 20 and 110 kHz, and by (iii) variation of the static magnetic field B0. The experimental SNR values are validated with numerical simulations employing up to 9 proton spins. Although in reality a protein would contain far more than 9 protons, in a deuterated environment this is a sufficient number to achieve satisfactory simulations consistent with the experimental data. The key results of this analysis are (i) with current hardware, deuteration is still necessary to record spectra of optimum quality; (ii) 13CH3 isotopomers for methyl groups yield the best SNR when MAS frequencies above 100 kHz are available; and (iii) sensitivity increases with a factor beyond B0 3/2 with the static magnetic field due to a transition of proton-proton dipolar interactions from a strong to a weak coupling limit.

The periodic axon membrane skeleton leads to Na nanodomains but does not impact action potentials.

Recent work has established that axons have a periodic skeleton structure comprising of azimuthal actin rings connected via longitudinal spectrin tetramer filaments. This structure endows the axon with structural integrity and mechanical stability. Additionally, voltage-gated sodium channels follow the periodicity of the active-spectrin arrangement, spaced ∼190 nm segments apart. The impact of this periodic arrangement of sodium channels on the generation and propagation of action potentials is unknown. To address this question, we simulated an action potential using the Hodgkin-Huxley formalism in a cylindrical compartment, but instead of using a homogeneous distribution of voltage-gated sodium channels in the membrane, we applied the experimentally determined periodic arrangement. We found that the periodic distribution of voltage-gated sodium channels does not significantly affect the generation or propagation of action potentials but instead leads to large, localized sodium action currents caused by high-density sodium nanodomains. Additionally, our simulations show that the distance between periodic sodium channel strips could control axonal excitability, suggesting a previously underappreciated mechanism to regulate neuronal firing properties. Together, this work provides a critical new insight into the role of the periodic arrangement of sodium channels in axons, providing a foundation for future experimental studies.

Spectrins and human diseases.

Spectrin, as one of the major components of a plasma membrane-associated cytoskeleton, is a cytoskeletal protein composed of the modular structure of α and ß subunits. The spectrin-based skeleton is essential for preserving the integrity and mechanical characteristics of the cell membrane. Moreover, spectrin regulates a variety of cell processes including cell apoptosis, cell adhesion, cell spreading, and cell cycle. Dysfunction of spectrins is implicated in various human diseases including hemolytic anemia, neurodegenerative diseases, ataxia, heart diseases, and cancers. Here, we briefly discuss spectrins function as well as the clinical manifestations and currently known molecular mechanisms of human diseases related to spectrins, highlighting that strategies for targeting regulation of spectrins function may provide new avenues for therapeutic intervention for these diseases.

An alpha II spectrin mutant peptide with unstable scaffold structure and increased sensitivity to calpain cleavage.

A spontaneous missense mutation in the alpha II spectrin (αII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD αII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of αII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the αII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant αII peptides, composed of spectrin repeat 9-10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 °C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound αII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration.

Optical control of fast and processive engineered myosins in vitro and in living cells.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

ßII spectrin (SPTBN1): biological function and clinical potential in cancer and other diseases.

ßII spectrin, the most common isoform of non-erythrocyte spectrin, is a cytoskeleton protein present in all nucleated cells. Interestingly, ßII spectrin is essential for the development of various organs such as nerve, epithelium, inner ear, liver and heart. The functions of ßII spectrin include not only establishing and maintaining the cell structure but also regulating a variety of cellular functions, such as cell apoptosis, cell adhesion, cell spreading and cell cycle regulation. Notably, ßII spectrin dysfunction is associated with embryonic lethality and the DNA damage response. More recently, the detection of altered ßII spectrin expression in tumors indicated that ßII spectrin might be involved in the development and progression of cancer. Its mutations and disorders could result in developmental disabilities and various diseases. The versatile roles of ßII spectrin in disease have been examined in an increasing number of studies; nonetheless, the exact mechanisms of ßII spectrin are still poorly understood. Thus, we summarize the structural features and biological roles of ßII spectrin and discuss its molecular mechanisms and functions in development, homeostasis, regeneration and differentiation. This review highlight the potential effects of ßII spectrin dysfunction in cancer and other diseases, outstanding questions for the future investigation of therapeutic targets. The investigation of the regulatory mechanism of ßII spectrin signal inactivation and recovery may bring hope for future therapy of related diseases.

Cotranslational folding cooperativity of contiguous domains of α-spectrin.

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

Impact of hydrodynamic interactions on protein folding rates depends on temperature.

We investigated the impact of hydrodynamic interactions (HI) on protein folding using a coarse-grained model. The extent of the impact of hydrodynamic interactions, whether it accelerates, retards, or has no effect on protein folding, has been controversial. Together with a theoretical framework of the energy landscape theory (ELT) for protein folding that describes the dynamics of the collective motion with a single reaction coordinate across a folding barrier, we compared the kinetic effects of HI on the folding rates of two protein models that use a chain of single beads with distinctive topologies: a 64-residue α/ß chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue ß-barrel α-spectrin Src-homology 3 domain (SH3) protein. When comparing the protein folding kinetics simulated with Brownian dynamics in the presence of HI to that in the absence of HI, we find that the effect of HI on protein folding appears to have a "crossover" behavior about the folding temperature. This means that at a temperature greater than the folding temperature, the enhanced friction from the hydrodynamic solvents between the beads in an unfolded configuration results in lowered folding rate; conversely, at a temperature lower than the folding temperature, HI accelerates folding by the backflow of solvent toward the folded configuration of a protein. Additionally, the extent of acceleration depends on the topology of a protein: for a protein like CI2, where its folding nucleus is rather diffuse in a transition state, HI channels the formation of contacts by favoring a major folding pathway in a complex free energy landscape, thus accelerating folding. For a protein like SH3, where its folding nucleus is already specific and less diffuse, HI matters less at a temperature lower than the folding temperature. Our findings provide further theoretical insight to protein folding kinetic experiments and simulations.

Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis.

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin­5­maleimide (EMA)­labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix­assisted laser desorption­ionization time of flight (MALDI­TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large­scale gene screening using high­resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS­PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA­labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531­12C>T. The corresponding mutant polypeptides were also analyzed by MALDI­TOF mass spectroscopy. SDS­PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression.

Structural analysis of P. falciparum KAHRP and PfEMP1 complexes with host erythrocyte spectrin suggests a model for cytoadherent knob protrusions.

Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) and Knob-associated Histidine-rich Protein (KAHRP) are directly linked to malaria pathology. PfEMP1 and KAHRP cluster on protrusions (knobs) on the P. falciparum-infected erythrocyte surface and enable pathogenic cytoadherence of infected erythrocytes to the host microvasculature, leading to restricted blood flow, oxygen deprivation and damage of tissues. Here we characterize the interactions of PfEMP1 and KAHRP with host erythrocyte spectrin using biophysical, structural and computational approaches. These interactions assist knob formation and, thus, promote cytoadherence. We show that the folded core of the PfEMP1 cytosolic domain interacts broadly with erythrocyte spectrin but shows weak, residue-specific preference for domain 17 of α spectrin, which is proximal to the erythrocyte cytoskeletal junction. In contrast, a protein sequence repeat region in KAHRP preferentially associates with domains 10-14 of ß spectrin, proximal to the spectrin-ankyrin complex. Structural models of PfEMP1 and KAHRP with spectrin combined with previous microscopy and protein interaction data suggest a model for knob architecture.

Effects of heat and freeze on isolated erythrocyte submembrane skeletons.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.

Whole Exome Sequencing Reveals the Order of Genetic Changes during Malignant Transformation and Metastasis in a Single Patient with NF1-plexiform Neurofibroma.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) occur at increased frequency in individuals with neurofibromatosis type 1 (NF1), where they likely arise from benign plexiform neurofibroma precursors. While previous studies have used a variety of discovery approaches to discover genes associated with MPNST pathogenesis, it is currently unclear what molecular events are associated with the evolution of MPNST from plexiform neurofibroma. EXPERIMENTAL DESIGN: Whole-exome sequencing was performed on biopsy materials representing plexiform neurofibroma (n = 3), MPNST, and metastasis from a single individual with NF1 over a 14-year period. Additional validation cases were used to assess candidate genes involved in malignant progression, while a murine MPNST model was used for functional analysis. RESULTS: There was an increasing proportion of cells with a somatic NF1 gene mutation as the tumors progressed from benign to malignant, suggesting a clonal process in MPNST development. Copy number variations, including loss of one copy of the TP53 gene, were identified in the primary tumor and the metastatic lesion, but not in benign precursor lesions. A limited number of genes with nonsynonymous somatic mutations (ßIII-spectrin and ZNF208) were discovered, several of which were validated in additional primary and metastatic MPNST samples. Finally, increased ßIII-spectrin expression was observed in the majority of MPNSTs, and shRNA-mediated knockdown reduced murine MPNST growth in vivo. CONCLUSIONS: Collectively, the ability to track the molecular evolution of MPNST in a single individual with NF1 offers new insights into the sequence of genetic events important for disease pathogenesis and progression for future mechanistic study.

[Disallowed conformations of polypeptide chain exemplified by the ß-bend of the ß-hairpin in the α-spectrin CH3-domain].

The work presents the results of an exhaustive conformational analysis of ß-turns involving amino acid residues with disallowed backbone conformation of the polypeptide chain. It is known that the first residue of the ß-turn (Asn47) of the distal ß-hairpin in the α-spectrin SH3-domain is characterized by sterically disallowed main chain conformation (values of the dihedral angles (φ and ψ are in the right bottom quadrant of the Ramachandran plot). All α-spectrin structures with the anomalous elements deposited in the PDB were analysed. We hypothesized that the formation of disallowed conformation may occur through the fixation (due to the SH3 domain structure) of the adjacent to the ß-turn amino acid residues with the ß-structure. These residues are disposed in such a manner that ß-turn conformation of the residues contributes just to the disallowed local conformation of this residue whereas any other ß-turn conformations (with allowed local conformation) are impossible. To test this hypothesis an exhaustive conformational analysis of the ß-bend has been performed by altering internal coordinates (two pairs of φ and ψ angles and two Ω angles). The conformations were selected as a result of grid search procedure with. 1 degrees step that corresponded to stereochemically allowed local deformations of the polypeptide chain segment forming the ß-turn. In all conformations obtained the local conformation of Asn47 rests in the disallowed region. The conformations found include conformations coinciding with experimentally determined structures from the PDB as well as an additional variant that differs from X-ray structure in values of a pair of φ and ψ angles of the second residue belonging to the ß-bend. Values of these angles fall in the region of the Ramachandran plot near the line φ = 0 (and negative values of ψ) i.e. in strongly disallowed region without experimental points. Therefore the additional variants of the ß-turn local deformation are impossible to observe in experiment. Thus, the idea that disallowed conformation is intruded to the ß-bend by fixation of adjacent residues receives confirmation in this work. The topological limitations in a context of the structure in such kind of ß-hairpins exclude the allowed local conformations.

Spectrin binding motifs regulate Scribble cortical dynamics and polarity function.

The tumor suppressor protein Scribble (SCRIB) plays an evolutionary conserved role in cell polarity. Despite being central for its function, the molecular basis of SCRIB recruitment and stabilization at the cell cortex is poorly understood. Here we show that SCRIB binds directly to the CH1 domain of ß spectrins, a molecular scaffold that contributes to the cortical actin cytoskeleton and connects it to the plasma membrane. We have identified a short evolutionary conserved peptide motif named SADH motif (SCRIB ABLIMs DMTN Homology) which is necessary and sufficient to mediate protein interaction with ß spectrins. The SADH domains contribute to SCRIB dynamics at the cell cortex and SCRIB polarity function. Furthermore, mutations in SCRIB SADH domains associated with spina bifida and cancer impact the stability of SCRIB at the plasma membrane, suggesting that SADH domain alterations may participate in human pathology.

Spectrin: structure, function and disease.

Spectrin is a large, cytoskeletal, and heterodimeric protein composed of modular structure of α and ß subunits, it typically contains 106 contiguous amino acid sequence motifs called "spectrin repeats". Spectrin is crucial for maintaining the stability and structure of the cell membrane and the shape of a cell. Moreover, it contributes to diverse cell functions such as cell adhesion, cell spreading, and the cell cycle. Mutations of spectrin lead to various human diseases such as hereditary hemolytic anemia, type 5 spinocerebellar ataxia, cancer, as well as others. This review focuses on recent advances in determining the structure and function of spectrin as well as its role in disease.

Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates.

Spectrin and ankyrin are membrane skeletal proteins that contribute to mechanical support of plasma membranes and micron-scale organization of diverse membrane-spanning proteins. This chapter provides a plausible scenario for the evolution of ankyrin- and spectrin-based membrane domains with a focus on vertebrates. The analysis integrates recent phylogenetic information with functional analyses of spectrin and ankyrin in erythrocytes, axon initial segments and nodes of Ranvier in neurons, T-tubules and intercalated disks of cardiomyocytes, lateral membrane domains of epithelial cells, and costameres of striated muscle. A core spectrin-ankyrin mechanism for coordinating membrane-spanning proteins and mechanically stabilizing membrane bilayers was expanded in vertebrates by gene duplication events, insertion of giant alternately spliced exons of axonal ankyrins, and a versatile peptide-binding fold of ANK repeats that facilitated acquisition of new protein partners. Cell adhesion molecules (CAM), including dystroglycan, L1 CAM family members, and cadherins, are the earliest examples of membrane-spanning proteins with ankyrin-binding motifs and were all present in urochordates. In contrast, ion channels have continued to evolve ankyrin-binding sites in vertebrates. These considerations suggest a model where proto-domains formed through interaction of ankyrin and spectrin with CAMs. These proto-domains then became populated with ion channels that developed ankyrin-binding activity with selective pressure provided by optimization of physiological function. The best example is the axon initial segment where ankyrin-binding activity evolved sequentially and independently first in L1 CAMs, then in voltage-gated sodium channels, and finally in KCNQ2/3 channels, with the selective advantage of fast and precisely regulated signaling.

Spectrin domain of eukaryotic initiation factor 3a is the docking site for formation of the a:b:i:g subcomplex.

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10-15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.

High-resolution crystal structure of spectrin SH3 domain fused with a proline-rich peptide.

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the two-fold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.