The A-type lamins (lamin A/C), encoded by the LMNA gene, are important structural components of the nuclear lamina. LMNA mutations lead to degenerative disorders known as laminopathies, including the premature aging disease Hutchinson-Gilford progeria syndrome. In addition, altered lamin A/C expression is found in various cancers. Reports indicate that lamin A/C plays a role in DNA double strand break repair, but a role in DNA base excision repair (BER) has not been described. We provide evidence for reduced BER efficiency in lamin A/C-depleted cells (Lmna null MEFs and lamin A/C-knockdown U2OS). The mechanism involves impairment of the APE1 and POLβ BER activities, partly effectuated by associated reduction in poly-ADP-ribose chain formation. Also, Lmna null MEFs displayed reduced expression of several core BER enzymes (PARP1, LIG3 and POLβ). Absence of Lmna led to accumulation of 8-oxoguanine (8-oxoG) lesions, and to an increased frequency of substitution mutations induced by chronic oxidative stress including GC>TA transversions (a fingerprint of 8-oxoG:A mismatches). Collectively, our results provide novel insights into the functional interplay between the nuclear lamina and cellular defenses against oxidative DNA damage, with implications for cancer and aging.

The Italian Society for Pure and Applied Biophysics (SIBPA) held its XXIV National Congress in the beautiful seaside town of Ancona, Italy, on September 10-13, 2018. This special issue features a selection of contributions from the Congress in all areas of modern biophysics including molecular, cellular, applied, computational and nanoscale biophysics. SIBPA pursues its institutional tasks and carries on its successful promotion of biophysical disciplines at the national and international levels, also trough the consolidation of its partnership with Biophysical Chemistry and Elsevier.

There is an abundance of RNA sequence information available due to the efforts of sequencing projects. However, current techniques implemented to solve the tertiary structures of RNA, such as NMR and X-ray crystallography, are difficult and time-consuming. Therefore, biophysical techniques are not able to keep pace with the abundance of sequence information available. Because of this, there is a need to develop quick and efficient ways to predict RNA tertiary structure from sequence. One promising approach is to identify structural patterns within previously solved 3D structures and apply these patterns to new sequences. RNA tetraloops are one of the most common naturally occurring secondary structure motifs. Here, we use RNA Characterization of Secondary Structure Motifs (CoSSMos), Dissecting the Spatial Structure of RNA (DSSR), and a bioinformatic approach to search for and characterize tertiary structure patterns among tetraloops. Not surprising, we identified the well-known GNRA and UNCG tetraloops, as well as the previously identified RNYA tetraloop. However, some previously identified characteristics of these families were not observed in this data set, and some new characteristics were identified. In addition, we also identified and characterized three new tetraloop sequence families: YGAR, UGGU, and RMSA. This new structural information sheds light on the tertiary structure of tetraloops and contributes to the efforts of RNA tertiary structure prediction from sequence.

Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na binding, reduces stability of the transition state, collapses the 215-217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na binding and the E*-E equilibrium.

Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can partially prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in 2 mouse models of kidney disease. These effects were not related to changes in circulating leukocytes because bone marrow transplants from miR-33-deficient animals did not have a similar impact on disease progression. Most important, targeted delivery of miR-33 peptide nucleic acid inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of and genes within the operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered 'SufFeScient' derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

Following publication of the original article [1], it was reported that Fig. 1c was not entirely readable due to overlapping Fig. 1d. The publishers apologise for this error.

Metabolic reprogramming is one of the hallmarks of cancer which favours rapid energy production, biosynthetic capabilities and therapy resistance. In our previous study, we showed bitter melon extract (BME) prevents carcinogen induced mouse oral cancer. RNA sequence analysis from mouse tongue revealed a significant modulation in "Metabolic Process" by altering glycolysis and lipid metabolic pathways in BME fed group as compared to cancer group. In present study, we evaluated the effect of BME on glycolysis and lipid metabolism pathways in human oral cancer cells.