Costain 1 (ARM19800.1) - The first identified protein of the costa of the pathogenic protozoan Tritrichomonas foetus.

Protists members of the Trichomonadidae and Tritrichomonadidae families include agents of trichomoniasis that constitute important parasitic diseases in humans and in animals of veterinary interest. One of the characteristic features of these eukaryotic microorganisms is that they contain a fibrous structure known as the costa as an important cytoskeleton structure, that differs in several aspects from other cytoskeleton structures found in eukaryotic cells. Previous proteomic analysis of an enriched costa fraction revealed the presence of several hypothetical proteins. Here we describe the localization of one of the most prevalent protein found in this previously made proteomic assay to confirm its presence in the costa of Tritrichomonas foetus. A peptide sequence of the hypothetical protein ARM19800.1 was selected for the production of specific polyclonal antibodies and its specificity was confirmed by Western Blotting using an enriched costa fraction. Next, the specific localization of the selected protein was evaluated by immunofluorescence and electron microscopy immunocytochemistry. Our observations clearly showed that the ARM 19800.1 protein is indeed localized in the costa and displays an almost periodic labeling pattern. Since this is the first protein identified in the costa, it was designated as costain 1. A better understanding of a structure as peculiar as the costa is of great biological and evolutionary importance due to the fact that it contains unique proteins, it may represent a possible chemotherapy target and it may correspond to antigens of interest in immunodiagnosis and/or vaccine development.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium.

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly.

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19cCCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.

Microtubule Attachment and Centromeric Tension Shape the Protein Architecture of the Human Kinetochore.

The nanoscale protein architecture of the kinetochore plays an integral role in specifying the mechanisms underlying its functions in chromosome segregation. However, defining this architecture in human cells remains challenging because of the large size and compositional complexity of the kinetochore. Here, we use Förster resonance energy transfer to reveal the architecture of individual kinetochore-microtubule attachments in human cells. We find that the microtubule-binding domains of the Ndc80 complex cluster at the microtubule plus end. This clustering occurs only after microtubule attachment, and it increases proportionally with centromeric tension. Surprisingly, Ndc80 complex clustering is independent of the organization and number of its centromeric receptors. Moreover, this clustering is similar in yeast and human kinetochores despite significant differences in their centromeric organizations. These and other data suggest that the microtubule-binding interface of the human kinetochore behaves like a flexible "lawn" despite being nucleated by repeating biochemical subunits.

Anatomic characteristics of epididymis based on histology, proteomic, and 3D reconstruction.

BACKGROUND: The epididymis is a popular research topic in urology and reproduction. OBJECTIVES: To explore and identify the anatomical characteristics of the epididymis based on histology, proteomics, and 3D reconstruction of epididymal tubules. MATERIALS AND METHODS: A 3D reconstruction of epididymal tubules was generated based on 7-µm-thick transverse serial sections of an epididymis. The proteins in the subcompartments of the epididymis were obtained and analyzed by non-labeled sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Protein function, signaling pathways, protein expression, and the histology in different subcompartments were analyzed. RESULTS: The caput (Cap), corpus (Cor), and cauda (Cau) of the epididymis were divided into 6, 10, and 4 subcompartments, respectively, and the subcompartment between the Cap and Cor is mixed together. A total of 3411 proteins were identified, and 854 proteins were accurately quantified after screening. When the subcompartment Cap 5 transitioned to Cap 6 and Cap 6 to Cap 7, 87 and 52 proteins were upregulated and 14 and 7 proteins were downregulated, respectively. The Cor 9 transition to Cau 1 was marked by 230 proteins that were downregulated, while 74 proteins were upregulated. At the junction of the cauda and the vas deferens, 57 proteins were downregulated, and 410 proteins were upregulated. Cap 6 histology was consistent with that of Cor 1. DISCUSSION AND CONCLUSION: The epididymis contains distinct connective tissue septa that can be identified under a surgical tabletop microscope, enabling it to be divided into 20 subcompartments.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.

Identification and characterization of novel filament-forming proteins in cyanobacteria.

Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD+, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 ± 2000 M-1 s-1) is at least as active as the TIR domain alone (kcat/KM = 1500 ± 300 M-1 s-1). Finally, we show that the SARM1 hydrolyzes NAD+ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.

Expression, purification, and structural analysis of the full-length human integral membrane protein γ-sarcoglycan.

Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, this data allows for opportunities in the future for enhanced treatment modalities and cures for LGMD.

Cytoskeletal and extracellular matrix proteins resist the burning of bones.

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.

Structure of BAI1/ELMO2 complex reveals an action mechanism of adhesion GPCRs via ELMO family scaffolds.

The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G protein-coupled receptors (aGPCRs) plays crucial roles in diverse cellular processes including phagocytosis, myoblast fusion, and synaptic development through the ELMO/DOCK/Rac signaling pathway, although the underlying molecular mechanism is not well understood. Here, we demonstrate that an evolutionarily conserved fragment located in the C-terminal cytoplasmic tail of BAI-aGPCRs is specifically recognized by the RBD-ARR-ELMO (RAE) supramodule of the ELMO family scaffolds. The crystal structures of ELMO2-RAE and its complex with BAI1 uncover the molecular basis of BAI/ELMO interactions. Based on the complex structure we identify aGPCR-GPR128 as another upstream receptor for the ELMO family scaffolds, most likely with a recognition mode similar to that of BAI/ELMO interactions. Finally, we map disease-causing mutations of BAI and ELMO and analyze their effects on complex formation.

Purification and characterization of FtsZ from the citrus canker pathogen Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xac) is the causative agent of citrus canker, a plant disease that significantly impacts citriculture. In earlier work, we showed that alkylated derivatives of gallic acid have antibacterial action against Xac and target both the cell division protein FtsZ and membrane integrity in Bacillus subtilis. Here, we have purified native XacFtsZ and characterized its GTP hydrolysis and polymerization properties. In a surprising manner, inhibition of XacFtsZ activity by alkyl gallates is not as strong as observed earlier with B. subtilis FtsZ. As the alkyl gallates efficiently permeabilize Xac membranes, we propose that this is the primary mode of antibacterial action of these compounds.

Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics.

Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.

Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia.

To study prokaryotic expression and subcellular localization of α-13 giardin in Giardia lamblia trophozoites, α-13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α-13 giardin was determined by fluorescence immunoassay. The results showed that the length of α-13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti-α13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α-13 giardin. Immunofluorescence assays revealed that α-13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α-13 giardin of G. lamblia.

Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last 10% of the cell cycle.

Bacterial cell division is a highly regulated process, which involves the formation of a complex apparatus, the divisome, by over a dozen proteins. In the few model bacteria in which the division process was detailed, divisome assembly occurs in two distinct steps: a few proteins, including the FtsZ tubulin-like protein, form a membrane associated contractile ring, the Z-ring, at ~30% of the cell cycle. The Z-ring serves as a scaffold for the recruitment of a second series of proteins, including integral membrane and periplasmic cell wall remodelling enzymes, at ~50% of the cell cycle. Actual septation occupies most of the remaining half of the cell cycle. In contrast, we present evidence suggesting that early pre-divisional Z-rings form between 40 and 50% of the cell cycle and mature into fully assembled divisome at about 80% of the cell cycle in Vibrio cholerae. Thus, actual septation is restricted to a very short amount of time. Our results further suggest that late assembly of the divisome probably helps maintain the asymmetric polar organisation of V. cholerae cells by limiting the accumulation of a cell pole marker, HubP, at the nascent cell poles.

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.

Studying the Mechanochemistry of Processive Cytoskeletal Motors With an Optical Trap.

Cytoskeletal motors utilize the energy stored in ATP to generate linear motion along rigid filaments. Because their enzymatic cycles are tightly coupled to the production of force and forward movement, the optical-trapping technique is uniquely suited for studying their mechanochemical cycle. Here, we discuss the practical aspects of optical trapping in connection with single-motor assays and describe three distinct experimental modes (fixed-trap, force feedback, and square wave) that are typically used to investigate the enzymatic and biophysical properties of cytoskeletal motors. The principal outstanding questions in the field involve motor regulation by cargo adaptor proteins and cargo transport by teams of motors, ensuring that the optical trap's ability to apply precise forces and measure nanometer-scale displacements will remain crucial to the study of intracellular motility in the foreseeable future.

In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP.

Tem1 is a small GTPase that controls the mitotic progression of Saccharomyces cerevisiae through the Mitotic Exit Network. Tem1 activity is tightly controlled in mitosis by Bub2 and Bfa1 and is also regulated by the spindle orientation checkpoint that monitors the correct alignment of the mitotic spindle with the mother-daughter axis. In this chapter we describe the purification of Tem1, Bfa1, and Bub2 and a detailed radioactive filter-binding assay to study the nucleotide binding properties of Tem1 and the role of its regulators Bfa1 and Bub2.

Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.

Purification and Structural Analysis of Plectin and BPAG1e.

Plectin and BPAG1e belong to the plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions. Owing to the mosaic organization of plakins, the structure of their constituent individual domains or small multi-domain segments can be analyzed isolated. Yet, understanding the integrated function of large regions, oligomers, and heterocomplexes of plakins is difficult due to the large and segmented structure. Here, we describe methods for the production of plectin and BPAG1e samples suitable for structural and biophysical analysis. In addition, we discuss the combination of hybrid methods that yield information at several resolution levels to study the complex, multi-domain, and flexible structure of plakins.