Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides

The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. On the basis of genetic evidence, it has been postulated that the FHIT protein may function as tumor suppressor, implying a role for the FHIT protein in carcinogenesis. Recently, Gaudio et al. reported that FHIT binds and delocalizes annexin A4 (ANXA4) from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. They also identified the smallest protein sequence of the FHIT still interacting with ANXA4, ranging from position 7 to 13: QHLIKPS. This short sequence of FHIT protein was not only able to bind ANXA4 but also to hold its target in the cytosol during paclitaxel treatment, thus avoiding ANXA4 translocation to the inner side of the cell membrane. Starting from these results, to obtain much information about structure requirements involved in the interaction of the peptide mentioned above, we synthetized a panel of seven peptides through an Ala-scan approach. In detail, to study the binding of FHIT derived peptides with ANXA4, we applied a combination of different biophysical techniques such as differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). Circular dichroism (CD) and nuclear magnetic resonance (NMR) were used to determine the conformational structure of the lead peptide (7–13) and peptides generated from ala-scan technique. The application of different biophysical and structural techniques, integrated by a preliminary biological evaluation, allowed us to build a solid structure activity relationship on the synthesized peptides.

Annexin A4 Is Dispensable for Hair Cell Development and Function

Annexin A4 (ANXA4) is a Ca2+-dependent phospholipid-binding protein that is specifically expressed in the cochlear and vestibular hair cells, but its function in the hair cells remains unknown. In the present study, we show that besides localizing on the plasma membrane, ANXA4 immunoreactivity is also localized at the tips of stereocilia in the hair cells. In order to investigate the role of ANXA4 in the hair cells, we established Anxa4 knockout mice using CRISPR/Cas9 technique. Unexpectedly, the development of both cochlear and vestibular hair cells is normal in Anxa4 knockout mice. Moreover, stereocilia morphology of Anxa4 knockout mice is normal, so is the mechano-electrical transduction (MET) function. Consistently, the auditory and vestibular functions are normal in the knockout mice. In conclusion, we show here that ANXA4 is dispensable for the development and function of hair cells, which might result from functional redundancy between ANXA4 and other annexin(s) in the hair cells.

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles

Protein structure and curvature sensing for annexin A4 trimers are coupled. These findings may provide new insight for the mechanisms underlying plasma membrane repair.

Annexins Bend Wound Edges during Plasma Membrane Repair

The plasma membrane of eukaryotic cells defines the boundary to the extracellular environment and, thus provides essential protection from the surroundings. Consequently, disruptions to the cell membrane triggered by excessive mechanical or biochemical stresses pose fatal threats to cells, which they need to cope with to survive. Eukaryotic cells cope with these threats by activating their plasma membrane repair system, which is shared by other cellular functions, and includes mechanisms to remove damaged membrane by internalization (endocytosis), shedding, reorganization of cytoskeleton and membrane fusion events to reseal the membrane. Members of the annexin protein family, which are characterized by their Ca2+-dependent binding to anionic phospholipids, are important regulators of plasma membrane repair. Recent studies based on cellular and biophysical membrane models show that they have more distinct functions in the repair response than previously assumed by regulating membrane curvature and excision of damaged membrane. In cells, plasma membrane injury and flux of Ca2+ ions into the cytoplasm trigger recruitment of annexins including annexin A4 and A6 to the membrane wound edges. Here, they induce curvature and constriction force, which help pull the wound edges together for eventual fusion. Cancer cells are dependent on efficient plasma membrane repair to counteract frequent stress-induced membrane injuries, which opens novel avenues to target cancer cells through their membrane repair system. Here, we discuss mechanisms of single cell wound healing implicating annexin proteins and membrane curvature.