Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis.

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation.

Evidence for amylose inclusion complexes with multiple acyl chain lipids using solid-state NMR and theoretical approaches.

Amylose is known to form inclusion complexes in the presence of hydrophobic guests. Among lipids, only single-chain fatty acids have been reported as possible guests with the surrounding amylose in a well-defined V-helix conformation. Using experimental 13C solid-state NMR, we studied the formation of inclusion complexes between amylose and a variety of multiple-chains lipids of increasing complexity. Molecular dynamics simulations and calculations of 13C isotropic chemical shifts using the Density Functional Theory approach were performed to support the interpretation of experimental spectra. We provide unambiguous evidences that amylose forms inclusion complexes with lipids bearing multiple acyl chains. Amylose conformations around these lipids are characterized by {ϕ,ψ} anomeric bond dihedral angles near {115°,105°}. In the 13C NMR spectra, this translates into C1 and C4 chemical shifts of 102.5 ppm and 81.1 ppm, regardless of the helical conformation of the amylose surrounding the acyl chains.

Diversity of the Hydrogen Bond Network and Its Impact on NMR Parameters of Amylose B Polymorph: A Study Using Molecular Dynamics and DFT Calculations Within Periodic Boundary Conditions.

Classical molecular dynamics simulations have been combined with quantum (DFT) calculations of 13C NMR parameters in order to relate the experimental spectrum of the double-helix form of the amylose B-polymorph in highly crystalline conditions not only to its 3D structure but also to the arrangement of atoms in the crystal lattice. Structures obtained from these simulations or from geometry optimization procedures at the DFT level have shown the presence of hydrogen bond networks between sugars of the same helix or between residues of the two chains of the double helix. 13C NMR parameter calculations have revealed the impact of such a network on the chemical shifts of carbon atoms. In addition, DFT calculations using periodic boundary conditions were compulsory to highlight the presence of two types of sugar within the crystal sample. It allows us to confirm, theoretically, the experimental hypothesis that the existence of two distinct sugar types in the NMR spectrum is a consequence of crystal packing.

Structures and NMR spectra of short amylose-lipid complexes. Insight using molecular dynamics and DFT quantum chemical calculations.

It is well established that amylose folds in a helix conformation in presence of lipids. Structural features of such molecular complexes are often analysed using 13C NMR spectroscopy. The large size of amylose used to make such analysis doesn't allow to unambiguously correlate structure of polymers and spectroscopic signals. We present structural analysis of small sized amyloses complexed to palmitic acid using classical molecular dynamics. 15 glucoses residues are necessary for the amylose to fold around the palmitic acid in a well-established helix conformation. Simulating 13C NMR spectra using quantum chemical DFT approach, we demonstrate that these spectra are affected by amylose size and specific intramolecular hydrogen bonds. By mean of theoretical NMR spectra of a 19-residues amylose, we precise the attribution of each characteristic resonances. One chemical shift that is usually attributed to a specific carbon may be related to the existence of different inter or intramolecular hydrogen bonds.