The tumor suppressor protein p53, a transcription product of the anti-oncogene
TP53
, is a critical factor in preventing cellular cancerization and killing cancer cells by inducing apoptosis. As a ...result, p53 is often referred to as the “guardian of the genome.” Almost half of cancers possess genetic mutations in the
TP53
gene, and most of these mutations result in the malfunction of p53, which promotes aggregation. In some cases, the product of the
TP53
mutant allele shows higher aggregation propensity; the mutant co-aggregates with the normal (functional) p53 protein, thus losing cellular activity of the p53 guardian. Cancer might also progress because of the proteolytic degradation of p53 by activated E3 ubiquitination enzymes, MDM2 and MDM4. The inhibition of the specific interaction between MDM2 (MDM4) and p53 also results in increased p53 activity in cancer cells. Although the molecular targets of the drugs are different, two drug discovery strategies with a common goal, “rescuing p53 protein,” have recently emerged. To conduct this approach, various biophysical methods of protein characterization were employed. In this review, we focus on these two independent strategies based on the unique biophysical features of the p53 protein.
An increasing number of proteins, which have neither regular secondary nor well-defined tertiary structures, have been found to be present in cells. The structure of these proteins is highly flexible ...and disordered under physiological (native) conditions, and they are called “intrinsically disordered” proteins (IDPs). Many of the IDPs are involved in interactions with other biomolecules such as DNA, RNA, carbohydrates, and proteins. While these IDPs are largely unstructured by themselves, marked conformational changes often occur upon binding to an interacting partner, which is known as the “coupled folding and binding mechanism”, which enable them to change the conformation to become compatible with the shape of the multiple target biomolecules. We have studied the structure and interaction of eukaryotic transcription factors Sp1 and TAF4, and found that both of them have long intrinsically disordered regions (IDRs). One of the IDRs in Sp1 exhibited homo-oligomer formation. In addition, the same region was used for the interaction with another IDR found in the TAF4 molecule. In both cases, we have not detected any significant conformational change in that region, suggesting a prominent and novel binding mode for IDPs/IDRs, which are not categorized by the well-accepted concept of the coupled folding and binding mechanism.
The expression of eukaryotic genes is precisely controlled by interactions between general transcriptional factors and promoter‐specific transcriptional activators. The fourth element of TATA‐box ...binding protein‐associated factor (TAF4), an essential subunit of the general transcription factor TFIID, serves as a coactivator for various promoter‐specific transcriptional regulators. Interactions between TAF4 and site‐specific transcriptional activators, such as Sp1, are important for regulating the expression levels of genes of interest. However, only limited information is available on the molecular mechanisms underlying the interactions between these transcriptional regulatory proteins. We herein analyzed the interaction between the transcriptional factors Sp1 and TAF4 using high‐resolution solution nuclear magnetic resonance spectroscopy. We found that four glutamine‐rich (Q‐rich) regions in TAF4 were largely disordered under nearly physiological conditions. Among them, the first Q‐rich region in TAF4 was essential for the interaction with another Q‐rich region in the Sp1 molecule, most of which was largely disordered. The residues responsible for this interaction were specific and highly localized in a defined region within a range of 20–30 residues. Nevertheless, a detailed analysis of 13C‐chemical shift values suggested that no significant conformational change occurred upon binding. These results indicate a prominent and exceptional binding mode for intrinsically disordered proteins other than the well‐accepted concept of “coupled folding and binding.”
The anti-oncogenic protein p53 is a transcription factor that prevents tumorigenesis by inducing gene repair proteins or apoptosis under DNA damage. Since the DNA-binding domain of p53 (p53C) is ...aggregation-prone, the anti-oncogenic function of p53 is often lost in cancer cells. This tendency is rather severe in some tumor-related p53 mutants, such as R175H. In this study, we examined the effect of salts, including KCl and sugars, on the aggregation of p53C by monitoring two distinct aggregates: amorphous-like and amyloid-like. The amorphous aggregates are detectable with 8-(phenylamino)-1-naphthalenesulfonic acid (ANS) fluorescence, whereas the amyloid aggregates are sensitive to thioflavin-T (ThT) fluorescence. We found that KCl inhibited the formation of amorphous aggregates but promoted the formation of amyloid aggregates in a p53C R175H mutant. The salts exhibited different effects against the wild-type and R175H mutants of p53C. However, the ratio of ANS/ThT fluorescence for the wild-type and R175H mutant remained constant. KCl also suppressed the structural transition and loss of the DNA-binding function of p53C. These observations indicate the existence of multiple steps of p53C aggregation, probably coupled with the dissociation of Zn. Notably, amorphous aggregates and amyloid aggregates have distinct properties that could be discriminated by various small additives upon aggregation.
The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene‐specific transcriptional activators. The general ...transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box‐binding protein (TBP) and multiple TBP‐associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter‐specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP‐associated factor TAF4. We herein report the structural details of the glutamine‐rich domains (Q‐domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q‐domains of Sp1 and four Q‐domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q‐domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well‐accepted concept of “coupled folding and binding.”
Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in ...the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia.
Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system.
Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neurofibromatosis type 2 (NF2), which encodes merlin (moesin-ezrin-radixin-like protein), belongs to the band 4.1, ezrin, radixin, moesin (FERM) domain-containing 4.1 superfamily. Merlin shares ...sequence homology with ERM proteins, is evolutionarily conserved, and acts as a tumour suppressor. Here, we describe the molecular functions of merlin from a biophysical point of view. We describe the structural basis for merlin regulatory mechanisms based on its interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) along with its interaction partners, and then describe its physiological functions in cell–cell adhesions. Elucidation of these merlin functions will lead to a clear understanding of its fundamental roles in cells and tissues.
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•Merlin plays a role in cell–cell adhesions and contact inhibition of proliferation.•Merlin changes its conformation upon PI(4,5)P2-binding.•Membrane-binding affinity can be determined by liposome cosedimentation/coflotation assays.•FRAP is applicable to quantify merlin dynamics on the membranes.•Merlin induces liquid-liquid phase separation.
On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication ...of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill’s Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.
This Commentary describes a call for submissions for the upcoming Special Issue focused on the research topics presented at the Australian Society of Biophysics (ASB) in 2020 and 2021. Submissions ...from past and present ASB members who could not attend these meetings are also welcome as contributions to this special issue.
Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the ...protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).
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•Solution structure of human stomatin SPFH domain is determined.•A cryptic phosphate-binding pocket was identified.•Stomatin SPFH domain can form a fibril-like assembly at a high concentration.•Phosphate ions promote formation of the fibril-like assembly.
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