Biomedical subjects

Bohumil Maco

Publications and source records attributed to Bohumil Maco.

9 recordsLinked to original sources

PubMed2025

RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii to enable parasite motility and invasion.

The conoid is a dynamic, tubulin-based structure conserved across the Apicomplexa that undergoes extrusion during egress, gliding motility, and invasion in Toxoplasma gondii. This organelle traverses the apical polar ring (APR) in response to calcium waves and plays a critical role in controlling parasite motility. While the actomyosin-dependent extrusion of the conoid is beginning to be elucidated, the mechanism by which it remains apically anchored to the APR is still unclear. RNG2, a protein localized to both the conoid and the APR, has emerged as a strong candidate for mediating this connection. Biochemical analysis revealed that RNG2 is an unstable protein, undergoing extensive proteolytic cleavage both in the parasite and in heterologous expression systems. Its biochemical properties, with the presence of large coiled-coil domains, likely facilitate the formation of concatenated assemblies, enabling RNG2 to serve as a dynamic and resilient bridge between the conoid and the APR. Using a combination of iterative ultrastructure expansion microscopy and immunoelectron microscopy, we confirmed the localization of RNG2 to the 22 tethering elements bridging the APR and the conoid. Conditional depletion of RNG2 led to the striking detachment of the intact conoid organelle from the APR, supporting an essential role for RNG2 as a tether. Cryo-electron tomography of conoid-less parasites revealed that, in the absence of RNG2, the apical vesicle remains anchored to the plasma membrane, while the rhoptries follow the detached conoid. Although RNG2 depletion only mildly reduces microneme secretion, the parasites are immotile and exhibit impaired rhoptry discharge, highlighting the critical role of proper conoid anchorage in motility and host cell invasion. Comprehensive mutagenesis of RNG2 identified distinct regions responsible for binding to the conoid and the APR, and demonstrated that the full-length, intact protein is essential for bridging these two structures and for its functional activity. Altogether, RNG2 emerges as a pivotal protein that ensures conoid functionality and coordination in Coccidia.

Toxoplasma↗

PubMed2006

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Casein Kinase I↗

PubMed2006

Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy.

To study the ultrastructure of nuclear pore complexes (NPCs), a wide spectrum of different electron microscopy (EM) or atomic force microscopy (AFM) techniques can be employed. The combination of these methods can reveal new insights into the structural and functional organization of this important supramolecular machine through which nucleocytoplasmic transport occurs. Negative staining, quick freezing/freeze-drying/rotary metal shadowing, embedding and thin sectioning, cryoelectron microscopy and tomography, scanning electron microscopy, or combination with immunolabeling techniques are tools for collecting data and information about the three-dimensional structure and architecture of the NPCs. AFM enables investigation of the functional dynamics of native NPCs under physiological conditions.

Animals↗

PubMed2005

Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis.

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1.GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.

Adaptor Protein Complex 2↗

PubMed2005

Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2.

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2'). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structure-function relationships of Ang1 with Tie2.

3T3-L1 Cells↗

PubMed2005

Poly-Ig tandems from I-band titin share extended domain arrangements irrespective of the distinct features of their modular constituents.

The cellular function of the giant protein titin in striated muscle is a major focus of scientific attention. Particularly, its role in passive mechanics has been extensively investigated. In strong contrast, the structural details of this filament are very poorly understood. To date, only a handful of atomic models from single domain components have become available and data on poly-constructs are limited to scarce SAXS analyses. In this study, we examine the molecular parameters of poly-Ig tandems from I-band titin relevant to muscle elasticity. We revisit conservation patterns in domain and linker sequences of I-band modules and interpret these in the light of available atomic structures of Ig domains from muscle proteins. The emphasis is placed on features expected to affect inter-domain arrangements. We examine the overall conformation of a 6Ig fragment, I65-I70, from the skeletal I-band of soleus titin using SAXS and electron microscopy approaches. The possible effect of highly conserved glutamate groups at the linkers as well as the ionic strength of the medium on the overall molecular parameters of this sample is investigated. Our findings indicate that poly-Ig tandems from I-band titin tend to adopt extended arrangements with low or moderate intrinsic flexibility, independently of the specific features of linkers or component Ig domains across constitutively- and differentially-expressed tandems. Linkers do not appear to operate as free hinges so that lateral association of Ig domains must occur infrequently in samples in solution, even that inter-domain sequences of 4-5 residues length would well accommodate such geometry. It can be expected that this principle is generally applicable to all Ig-tandems from I-band titin.

Amino Acid Motifs↗

PubMed2004

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits.

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.

Adenosine Triphosphate↗

PubMed2003

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart.

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.

Animals↗

PubMedDate not supplied

Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex.

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.

Animals↗