scholarly journals Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Emilie Ma ◽  
Pauline Dupaigne ◽  
Laurent Maloisel ◽  
Raphaël Guerois ◽  
Eric Le Cam ◽  
...  
Keyword(s):  
Current Model ◽  
Homology Search ◽  
Filament Formation ◽  
Dna Damaging Agents ◽  
Filament Assembly ◽  
Homologous Recombination Process ◽  
Key Steps

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

scholarly journals Functional insight into the role of Orc6 in septin complex filament formation in Drosophila

2015 ◽  
Vol 26 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Katarina Akhmetova ◽  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Origin Recognition Complex ◽  
Spatial Organization ◽  
Cell Cortex ◽  
Filament Formation ◽  
Gtp Binding ◽  
Complex Functions ◽  
Septin Filament

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments

1992 ◽  
Vol 102 (1) ◽  
pp. 31-41 ◽  
Author(s):  
P.D. Kouklis ◽  
P. Traub ◽  
S.D. Georgatos
Keyword(s):  
Consensus Sequence ◽  
Sequence Motif ◽  
Sequence Motifs ◽  
3T3 Cells ◽  
Filament Assembly ◽  
In Vitro Assembly ◽  
Vimentin Filaments

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil 1a’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.

scholarly journals Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

2007 ◽  
Vol 404 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Gary Flom ◽  
Robert H. Behal ◽  
Luke Rosen ◽  
Douglas G. Cole ◽  
Jill L. Johnson
Keyword(s):  
Current Model ◽  
Heat Shock Protein 90 ◽  
C Terminus ◽  
Client Proteins ◽  
Definition Of ◽  
Necessary And Sufficient ◽  
In Vitro Interaction

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

scholarly journals RAD-ical New Insights into RAD51 Regulation

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 629 ◽  
Author(s):  
Meghan R. Sullivan ◽  
Kara A. Bernstein
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Homology Search ◽  
Model Organisms ◽  
Filament Formation ◽  
Dna Double Strand Breaks ◽  
Rad51 Paralogs ◽  
Human Rad51

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

scholarly journals Endocannabinoid System and Tumour Microenvironment: New Intertwined Connections for Anticancer Approaches

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3396
Author(s):  
Marta Iozzo ◽  
Giovanna Sgrignani ◽  
Giuseppina Comito ◽  
Paola Chiarugi ◽  
Elisa Giannoni
Keyword(s):  
Tumour Response ◽  
Endocannabinoid System ◽  
Metastatic Potential ◽  
Tumour Microenvironment ◽  
Depth Analysis ◽  
Key Steps ◽  
Shed Light

The tumour microenvironment (TME) is now recognised as a hallmark of cancer, since tumour:stroma crosstalk supports the key steps of tumour growth and progression. The dynamic co-evolution of the tumour and stromal compartments may alter the surrounding microenvironment, including the composition in metabolites and signalling mediators. A growing number of evidence reports the involvement of the endocannabinoid system (ECS) in cancer. ECS is composed by a complex network of ligands, receptors, and enzymes, which act in synergy and contribute to several physiological but also pathological processes. Several in vitro and in vivo evidence show that ECS deregulation in cancer cells affects proliferation, migration, invasion, apoptosis, and metastatic potential. Although it is still an evolving research, recent experimental evidence also suggests that ECS can modulate the functional behaviour of several components of the TME, above all the immune cells, endothelial cells and stromal components. However, the role of ECS in the tumour:stroma interplay remains unclear and research in this area is particularly intriguing. This review aims to shed light on the latest relevant findings of the tumour response to ECS modulation, encouraging a more in-depth analysis in this field. Novel discoveries could be promising for novel anti-tumour approaches, targeting the microenvironmental components and the supportive tumour:stroma crosstalk, thereby hindering tumour development.

scholarly journals RAD-ical New Insights into RAD51 Regulation

2018 ◽  
Author(s):  
Meghan R. Sullivan ◽  
Kara A. Bernstein
Keyword(s):  
Genome Stability ◽  
Homology Search ◽  
Model Organisms ◽  
Filament Formation ◽  
Dna Double Strand Breaks ◽  
Current Perspective ◽  
Rad51 Paralogs ◽  
Human Rad51

Accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions and can be repaired using homologous recombination (HR). HR is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here we discuss the current perspective on the RAD51 paralogs in vivo and in vitro function and their relationship with cancer in vertebrate models.

scholarly journals The Activation of MEK1 by Enhanced Homodimerization Drives Tumorigenesis

2017 ◽  
Author(s):  
Jimin Yuan ◽  
Wan Hwa Ng ◽  
Zizi Tian ◽  
Jiajun Yap ◽  
Manuela Baccarini ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Differential Resistance ◽  
Erk Signaling ◽  
Activation Loop ◽  
Erk Activation ◽  
Pathway Activation ◽  
Key Steps

SummaryHyperactive RAS/RAF/MEK/ERK signaling has a well-defined role in cancer biology. Aberrant pathway activation occurs mostly upstream of MEK; however, MEK mutations are prevalent in some cancer subsets. Here we show that cancer-related MEK mutants can be classified as those activated by relieving the inhibitory role of helix A, and those with in-frame deletions of β3-αC loop, which exhibit differential resistance to MEK inhibitors in vitro and in vivo. The β3-αC loop deletions activate MEK1 through enhancing homodimerization that can drive intradimer cross-phosphorylation of activation loop. Further, we demonstrate that MEK1 dimerization is required both for its activation by RAF and for its catalytic activity towards ERK. Our study identifies a novel group of MEK mutants, illustrates some key steps in RAF/MEK/ERK activation, and has important implications for the design of therapies targeting hyperactive RAS/RAF/MEK/ERK signaling in cancers.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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