Targeted therapy in BRAF mutated aggressive papillary craniopharyngioma: a case report and overview of the literature.

BACKGROUND: Papillary craniopharyngiomas harbor the BRAF V600E mutation, which paves the way for using BRAF inhibitor molecules to treat tumors refractory to standard therapies. Single case reports confirmed the efficacy of targeted therapy. However, most reports were limited by the short follow-up. We describe the long-term course of a patient treated with dual-agent BRAF and MEK inhibitors and review the available literature. CASE REPORT: A 75-year-old male patient had recurrence of a papillary craniopharyngioma after transsphenoidal surgery and Gamma Knife radiosurgery. Review of the pathologic specimen confirmed the presence of the BRAF V600E mutation. Because of the few therapeutic options, we decided to initiate BRAF/MEK inhibitor combined therapy for six months. Rapid reduction of the tumor occurred, but three months after quitting combined medical therapy the tumor recurred. BRAF/MEK inhibitor therapy was resumed and the tumor again showed a marked reduction. The second course was maintained for 20 months and the tumor showed another recurrence within three months, which, again, responded to a third course of targeted therapy. CONCLUSIONS: Our study confirms the excellent response of papillary craniopharyngioma to combined BRAF and MEK inhibitors. However, rapid tumor recurrence is the rule when medical therapy is stopped. Resistance to a second and third course of targeted therapy did not occur, suggesting that tumor mutations affecting the response to drugs seems an uncommon event in papillary craniopharyngioma. The exact role of targeted therapy in the treatment algorithm of papillary craniopharyngiomas has still to be refined.

KCTD1 is a new modulator of the KCASH family of Hedgehog suppressors.

The Sonic Hedgehog (Hh) signal transduction pathway plays a critical role in many developmental processes and, when deregulated, may contribute to several cancers, including basal cell carcinoma, medulloblastoma, colorectal, prostate, and pancreatic cancer. In recent years, several Hh inhibitors have been developed, mainly acting on the Smo receptor. However, drug resistance due to Smo mutations or non-canonical Hh pathway activation highlights the need to identify further mechanisms of Hh pathway modulation. Among these, deacetylation of the Hh transcription factor Gli1 by the histone deacetylase HDAC1 increases Hh activity. On the other end, the KCASH family of oncosuppressors binds HDAC1, leading to its ubiquitination and subsequent proteasomal degradation, leaving Gli1 acetylated and not active. It was recently demonstrated that the potassium channel containing protein KCTD15 is able to interact with KCASH2 protein and stabilize it, enhancing its effect on HDAC1 and Hh pathway. KCTD15 and KCTD1 proteins share a high homology and are clustered in a specific KCTD subfamily. We characterize here KCTD1 role on the Hh pathway. Therefore, we demonstrated KCTD1 interaction with KCASH1 and KCASH2 proteins, and its role in their stabilization by reducing their ubiquitination and proteasome-mediated degradation. Consequently, KCTD1 expression reduces HDAC1 protein levels and Hh/Gli1 activity, inhibiting Hh dependent cell proliferation in Hh tumour cells. Furthermore, analysis of expression data on publicly available databases indicates that KCTD1 expression is reduced in Hh dependent MB samples, compared to normal cerebella, suggesting that KCTD1 may represent a new putative target for therapeutic approaches against Hh-dependent tumour.

Impact of deleterious variants in other genes beyond BRCA1/2 detected in breast/ovarian and pancreatic cancer patients by NGS-based multi-gene panel testing: looking over the hedge.

BACKGROUND: Hereditary breast cancer (BC), ovarian cancer (OC), and pancreatic cancer (PC) are the major BRCA-associated tumours. However, some BRCA1/2-wild-type (wt) patients with a strong personal and/or family history of cancer need a further genetic testing through a multi-gene panel containing other high- and moderate-risk susceptibility genes. PATIENTS AND METHODS: Our study was aimed to assess if some BC, OC, or PC patients should be offered multi-gene panel testing, based on well-defined criteria concerning their personal and/or family history of cancer, such as earliness of cancer onset, occurrence of multiple tumours, or presence of at least two or more affected first-degree relatives. For this purpose, 205 out of 915 BC, OC, or PC patients, resulted negative for BRCA1/2 and with significant personal and/or family history of cancer, were genetically tested for germline pathogenic or likely pathogenic variants (PVs/LPVs) in genes different from BRCA1/2. RESULTS: Our investigation revealed that 31 (15.1%) out of 205 patients harboured germline PVs/LPVs in no-BRCA genes, including PALB2, CHEK2, ATM, MUTYH, MSH2, and RAD51C. Interestingly, in the absence of an analysis conducted through multi-gene panel, a considerable percentage (15.1%) of PVs/LPVs would have been lost. CONCLUSIONS: Providing a multi-gene panel testing to BRCA1/2-wt BC/OC/PC patients with a strong personal and/or family history of cancer could significantly increase the detection rates of germline PVs/LPVs in other cancer predisposition genes beyond BRCA1/2. The use of a multi-gene panel testing could improve the inherited cancer risk estimation and clinical management of patients and unaffected family members.

Gamma knife radiosurgery in patients with Nelson's syndrome.

PURPOSE: Nelson's syndrome (NS) is a long-term complication of bilateral adrenalectomy in patients with Cushing's disease. The best therapeutic strategy in NS has not been well defined. Gamma knife radiosurgery (GKRS) is very effective to stop the growth of the pituitary adenoma, which is the main goal of the treatment of patients with NS. We report the largest series of patients with NS treated by GKRS at a single center. METHODS: The study was an observational, retrospective analysis of 28 consecutive patients with NS treated by GKRS in our department between 1995 and 2019. All patients had a growing ACTH-secreting pituitary adenoma. The main outcome of the study was to assess by the Kaplan-Meier method the risk of tumor progression after GKRS. RESULTS: The median follow-up after GKRS treatment was 98 months (IQR 61-155 months, range 7-250 months). Two patients (7.1%) had a recurrence of disease during follow-up. The 10-year progression-free survival was 91.7% (95% CI 80.5-100%). No patient had deterioration of visual function or oculomotor function after GKRS. New onset of hypogonadism and hypothyroidism occurred in 18.8% and 14.3% of the patients at risk. CONCLUSION: Our study confirms that GKRS may stop the tumor growth in the majority of patients with NS, even though very aggressive adenomas may ultimately escape this treatment. Safety of GKRS was good in our experience, but due attention must be paid to planning the distribution of radiation to critical structures, especially in patients previously treated by radiation.

Ubiquitin binds the amyloid ß peptide and interferes with its clearance pathways.

Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid ß peptide (Aß) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aß peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aß40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Aß peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aß (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/ß-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aß competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aß has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aß1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aß peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aß peptide in the malfunction of proteome maintenance occurring in AD.

Focusing on the functional characterization of the anserinase from Oreochromis niloticus.

Carnosine, anserine and homocarnosine are the three most representative compounds of the histidine dipeptides family, widely distributed in mammals in different amounts depending on the species and the tissue considered. Histidine dipeptides are mainly degraded by two different carnosinase homologues: a highly specific metal-ion dependent carnosinase (CN1) located in serum and brain and a non-specific cytosolic form (CN2). The hydrolysis of such dipeptides in prokaryotes and eukaryotes is also catalyzed by the anserinase (ANSN). Such naturally occurring dipeptides represent an interesting topic because they seem to have numerous biological roles such as potential neuroprotective and neurotransmitter functions in the brain and therefore ANSN results to be a very interesting target of study. We here report, for the first time, cloning, expression of ANSN from the fish Oreochromis niloticus both in a mammalian and in a prokaryotic system, in order to perform deep functional studies by enzymatic assays in the presence of different metals and substrates. Furthermore, by means of a mass spectrometry-based proteomic approach, we analysed protein sequence and the potential presence of post-translational modifications in the mammalian recombinant protein. Finally, a preliminary structural characterization was carried out on ANSN produced in Escherichia coli.

A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 ß-like: from in silico identification to experimental evidence.

BACKGROUND: Host defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 ß-like protein. METHODS: Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. RESULTS: GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages. CONCLUSION: Human GVF27 may offer significant advantages as leads for the design of human-specific therapeutics. GENERAL SIGNIFICANCE: Human cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.

Legal basis of the Advanced Therapies Regulation.

Advanced therapy medicinal products consist of gene therapy, somatic cell therapy and tissue engineered products. Due to their specific manufacturing process and mode of action these products require specially tailored legislation. With Regulation (EC) No. 1394/2007, these needs have been met. Definitions of gene therapy, somatic cell therapy and tissue engineered products were laid down. A new committee, the Committee for Advanced Therapies, was founded, special procedures such as the certification procedure for small- and medium-sized enterprises were established and the technical requirements for Marketing Authorisation Applications (quality, non-clinical and clinical) were revised.

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.

alpha,beta-poly(asparthylhydrazide)-glycidyltrimethylammonium chloride copolymers (PAHy-GTA): novel polymers with potential for DNA delivery.

Hydrophilic polycations form complexes when mixed with plasmids. Following functionalisation with glycidyltrimethylammonium chloride (GTA) alpha,beta-poly(asparthylhydrazide) (PAHy), a water-soluble synthetic macromolecule, becomes polycationic and potentially useful for systemic gene delivery. Initially the biocompatibility of PAHy and PAHy-GTA derivatives with different degrees of positive charge substitution were studied and it was shown that PAHy-GTA was neither haemolytic nor cytotoxicity up to 1 mg/ml. After intravenous injection (125)I-labelled PAHy-GTA derivative containing 46 mol% (PAHy-GTA(b)) of trimethylammonium groups did not accumulate in the liver (4.1+/-0.9% of the recovered dose after 1 h) but was subjected to renal excretion (45+/-21% of the recovered dose was in the kidneys after 1 h). PAHy-GTA formed complexes with DNA (gel retardation) and they protected against degradation by DNase II. Finally the ability of the PAHy-GTA(b) derivative to mediate the transfection of HepG2 cells using the marker gene beta-galactosidase was studied. The optimum plasmid/polymer mass ratio was examined in comparison to LipofectACE, Lipofectin and polyethylenimine.

A single point mutation (Glu85Arg) increases the stability of the thioredoxin from Escherichia coli.

Glu85 in the Escherichia coli thioredoxin, which is localized in the loop between beta4 and beta5, was substituted with the Arg present in the corresponding position in Bacillus acidocaldarius thioredoxin. This suggested that it could play an important role in the structure and thermostability of this protein owing to its involvement in numerous interactions. The effects of the mutation on the biophysical properties were analysed by circular dichroism, spectrofluorimetry and limited proteolysis, supported by molecular dynamics data. As modelling predicted, an increase in stability for E85R due to additional H-bonds between the beta5 and alpha4 regions was observed.

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three-dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H-NMR derived distance constraints along with 23 hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization. BacTrx consists of a well-defined core region of five strands of beta-sheet, surrounded by four exposed alpha-helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X-ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross-linking different secondary structural elements and clamping the C-terminal alpha-helix to the core of the protein.

Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability.

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.

Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach.

The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability. In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.

Oxidative modification of low-density lipoprotein and atherogenetic risk in beta-thalassemia.

We investigated the oxidative state of low-density lipoprotein (LDL) in patients with beta-thalassemia to determine whether there was an association with atherogenesis. Conjugated diene lipid hydroperoxides (CD) and the level of major lipid antioxidants in LDL, as well as modified LDL protein, were evaluated in 35 beta-thalassemia intermedia patients, aged 10 to 60, and compared with age-matched healthy controls. Vitamin E and beta-carotene levels in LDL from patients were 45% and 24% of that observed in healthy controls, respectively. In contrast, the mean amount of LDL-CD was threefold higher and lysil residues of apo B-100 were decreased by 17%. LDL-CD in thalassemia patients showed a strong inverse correlation with LDL vitamin E (r = -0.784; P <.0001), while a negative trend was observed with LDL-beta-carotene (r = -0.443; P =.149). In the plasma of thalassemia patients, malondialdehyde (MDA), a byproduct of lipid peroxidation, was increased by about twofold, while vitamin E showed a 52% decrease versus healthy controls. LDL-CD were inversely correlated with plasma vitamin E (r = -0.659; P <.0001) and correlated positively with plasma MDA (r = 0.621; P <. 0001). Plasma ferritin was positively correlated with LDL-CD (r = 0.583; P =.0002). No correlation was found between the age of the patients and plasma MDA or LDL-CD. The LDL from thalassemia patients was cytotoxic to cultured human fibroblasts and cytotoxicity increased with the content of lipid peroxidation products. Clinical evidence of mild to severe vascular complications in nine of the patients was then matched with levels of LDL-CD, which were 36% to 118% higher than the mean levels of the patients. Our results could account for the incidence of atherogenic vascular diseases often reported in beta-thalassemia patients. We suggest that the level of plasma MDA in beta-thalassemia patients may represent a sensitive index of the oxidative status of LDL in vivo and of its potential atherogenicity.

Oxidation resistance of LDL is correlated with vitamin E status in beta-thalassemia intermedia.

The alteration of the oxidant/antioxidant balance may affect the susceptibility of low density lipoproteins (LDL) to oxidation in haemolytic disorders such as thalassemia. Thirty patients affected by beta-thalassemia intermedia were examined, and compared with age-matched healthy controls. The mean amount of vitamin E in the thalassemic LDL was lower than control (p < 0.0001), either when it was calculated on the base of LDL protein (61% decrease) or cholesterol (25% decrease). The LDL resistance to Cu2+-induced oxidation, evaluated as the length of the lag phase before the onset of conjugated diene (CD) lipid hydroperoxide production, was 20% lower than control. Other parameters of LDL susceptibility to oxidation, such as the rate of lipid peroxidation, Rp, and the total amount of conjugated dienes produced, CDmax, were only slightly lower than control, which can be explained by a lower content of peroxidable lipids in the thalassemic LDL. Total LDL cholesterol was 1.08 x 10(3) and 2.07 x 10(3) mol/mol LDL in thalassemic and in control LDL, respectively. The length of the lag phase in thalassemic LDL shows a strongly positive correlation with its vitamin E content (r = 0.732; p < 0.0001). The r2-value of 0.53 provides evidence that more than 50% of the lag phase is determined by vitamin E. Oxidizability of LDL lipids may explain 22-24% of the lag phase, as calculated by the inverse correlation between the length of the lag phase and CDmax (r = -0.474; p = 0.008; r2 = 0.22) and Rp (r = -0.499; p = 0.005; r2 = 0.24). In multiple regression analysis, the lag phase was predictable to 66% by vitamin E plus CDmax, and to 60% by vitamin E plus Rp. Plasma vitamin E was 53% lower in thalassemia patients compared to control and positively correlated with vitamin E in the LDL (r = 0.677; p < 0.0001). None of the correlations above were observed in control subjects. In conclusion, beta-thalassemia is associated with very low levels of vitamin E in plasma and in LDL, a condition that renders these particles more susceptible to in vitro oxidative modification and may account for atherogenesis-related vascular diseases described in thalassemia. The present data on a statistically significant correlation between abnormally low vitamin E and oxidizability of LDL contribute substantially to the hypothesis that vitamin E is a pathophysiologically important determinant of antioxidative protection of LDL.

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Conformational studies of retro-inverso peptides: the crystal and molecular structure of the hydantoin from H-Ala-g-Ala-mGly-OBzl.

The crystal structure of the hydantoin 1-[(S)-1'-aminoethylmalonyl benzyl ester]-(S)-4-methylimidazolidin-2,5-dione (1) derived from the peptide H-Ala-gAla-mGly-OBzl, having the retro-inverso modification of the Ala-Gly bond, has been determined by x-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2(1) with a = 6.539, b = 14.721, c = 17.101 A, z = 4. The structure was solved by direct methods and refined with anisotropic thermal factors to a final R value of 0.067 for the 947 observed reflections. Reversal of the Ala-Gly amide bond perturbs the folding tendency of the backbone shown by the parent peptide t-BuCO-Ala-Gly-NHiPr. The gem-diamino residue, gAla, and the malonyl moieties are found in the helical and the extended conformations, respectively. Intramolecular hydrogen bonding is not observed. The molecules in the crystal are held together by the formation of two intermolecular hydrogen bonds of the N-H ... O=C type with N ... O distances of 2.86 and 3.17 A, respectively.