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Diacyl phosphatidylglycerols (PG) are the major anionic lipids in the Escherichia coli membrane. Short‐chain dihexanoyl phosphatidylglycerol (DHPG) was previously utilized for the structural determination, by NMR spectroscopy, of the peptide corresponding to the N‐terminal membrane anchor of the glucose‐specific enzyme IIA (IIAGlc) from E. coli. This study explores the possible use of lipid micelles of dioctanoyl phosphatidylglycerol (DOPG) and didecanoyl phosphatidylglycerol (DDPG) as alternatives to DHPG. At a peptide concentration of 1 mM, the minimum peptide/lipid molar ratios required for the formation of the lipid‐binding amphipathic helix are approximately 1 :40, 1 :5, and 1 :5 for DHPG, DOPG, and DDPG, respectively. Based on the lipid titration, the critical micelle concentration (CMC) of DHPG was estimated to be ∼50 mM. The 1H spectral linewidths of the peptide bound to a variety of lipid micelles decrease in the following order: DDPG > DOPG > DHPG. The helical regions of the peptide in different anionic lipids were elucidated based on chemical shift indexes (CSI). Residues Leu2‐Leu9, Leu2‐Val10, and Leu2‐Val10 were found to be helical in DHPG, DOPG, and DDPG, respectively, indicating that the lipid chain length had only a subtle effect on the amphipathic helix of the peptide. In light of the minimum peptide/lipid ratio and the spectral linewidth, and the CSI‐derived peptide structure, DOPG is proposed as a good compromise for structural studies of this membrane‐associated peptide by solution NMR spectroscopy. [ABSTRACT FROM AUTHOR]

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147–175). In the crystal structure of CC, the corresponding segment belongs to the α4 helix (residues 148–168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169–171) and a part of the α5 helix (171–175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize α-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20–160 μM) and low (2–16 μM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into α-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern–Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme. [Copyright &y& Elsevier]

Salmon calcitonin S-sulfonated analog (abbreviated as [S–SO3−]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500 IU/mg, which is about 30% higher than that (4500 IU/mg) of the wild type. The solution conformation of [S–SO3−]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic α-helix in the region of residues 4–22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO3−–Lys11+–Glu15−–Lys18+). The multiple salt bridges resulted in the stabilization of the longer amphipathic α-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic α-helix. [Copyright &y& Elsevier]

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300 , a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292 , lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292 , which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH. [ABSTRACT FROM AUTHOR]

Knowledge of the structural properties of linker histones is important to the understanding of their role in higher-order chromatin structure and gene regulation. Here we study the conformational properties of the peptide Ac-EKTPVKKKARKAAGGAKRKTSG-NH2 (NE-1) by circular dichroism and 1H-NMR. This peptide corresponds to the positively charged region of the N-terminal domain, adjacent to the globular domain, of mouse histone H1e (residues 15-36). This is the most abundant H1 subtype in many kinds of mammalian somatic cells. NE-1 is mainly unstructured in aqueous solution, but in the presence of the secondary-structure stabilizer trifluoroethanol (TFE) it acquires an α-helical structure. In 90% TFE solution the α-helical population is ∼40%. In these conditions, NE-1 is structured in two α-helices that comprise almost all the peptide, namely, from Thr17 to Ala27 and from Gly29 to Thr34. Both helical regions are highly amphipathic, with the basic residues on one face of the helix and the apolar ones on the other. The two helical elements are separated by a Gly-Gly motif. Gly-Gly motifs at equivalent positions are found in many vertebrate H1 subtypes. Structure calculations show that the Gly-Gly motif behaves as a flexible linker between the helical regions. The wide range of relative orientations of the helical axes allowed by the Gly-Gly motif may facilitate the tracking of the phosphate backbone by the helical elements or the simultaneous binding of two nonconsecutive DNA segments in chromatin. [ABSTRACT FROM AUTHOR]

A comprehensive phylogenetic analysis of the core subunits of succinate:quinone oxidoreductases and quinol:fumarate oxidoreductases is performed, showing that the classification of the enzymes as type A to E based on the type of the membrane anchor fully correlates with the specific characteristics of the two core subunits. A special emphasis is given to the type E enzymes, which have an atypical association to the membrane, possibly involving anchor subunits with amphipathic helices. Furthermore, the redox properties of the SQR/QFR proteins are also reviewed, stressing out the recent observation of redox-Bohr effect upon haem reduction, observed for the Desulfovibrio gigas and Rhodothermus marinus enzymes, which indicates a direct protonation event at the haems or at a nearby residue. Finally, the possible contribution of these enzymes to the formation/dissipation of a transmembrane proton gradient is discussed, considering recent experimental and structural data. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and, as such, recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have analyzed its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities., Competing Interests: RL is inventor on several patents related to the use of solnatide in edema reabsorption, ST, BF and HF are employees of Apeptico GmbH. The remaining authors declare no competing interests. Neither the EC nor the FFG had any role in the design of the research, collection, analysis, and interpretation of data or in writing the manuscript., (© 2022 The Author(s).)

Intrinsically disordered proteins and protein regions (IDPs/IDPRs) are mainly involved in signaling pathways, where fast regulation, temporal interactions, promiscuous interactions, and assemblies of structurally diverse components including membranes are essential. The autophagy pathway builds, de novo, a membrane organelle, the autophagosome, using carefully orchestrated interactions between proteins and lipid bilayers. Here, we discuss molecular mechanisms related to the protein disorder-based interactions of the autophagy machinery with membranes. We describe not only membrane binding phenomenon, but also examples of membrane remodeling processes including membrane tethering, bending, curvature sensing, and/or fragmentation of membrane organelles such as the endoplasmic reticulum, which is an important membrane source as well as cargo for autophagy. Summary of the current state of knowledge presented here will hopefully inspire new studies. A profound understanding of the autophagic protein-membrane interface is essential for advancements in therapeutic interventions against major human diseases, in which autophagy is involved including neurodegeneration, cancer as well as cardiovascular, metabolic, infectious, musculoskeletal, and other disorders.

Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane. [ABSTRACT FROM AUTHOR]

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor. [ABSTRACT FROM AUTHOR]

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2–8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome. [Display omitted] • WNK1 functions as an EMC assembly factor in a kinase-independent manner • WNK1 binds the soluble subunit EMC2 using a conserved amphipathic α-helix • WNK1 shields the hydrophobic EMC2–8 interface to prevent promiscuous interactions • WNK1 stabilizes unassembled EMC2 by competing for binding of E3 ubiquitin ligases Assembly of multi-subunit complexes is tightly regulated. The ER membrane protein complex (EMC) is an essential, nine-subunit membrane protein insertase. Pleiner et al. find that the kinase WNK1 is an assembly factor for the human EMC. WNK1 depletion perturbs EMC assembly and affects the biogenesis of its membrane protein clients. [ABSTRACT FROM AUTHOR]

What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding. [ABSTRACT FROM AUTHOR]

Amphipathic helical repeals arc considered as the structural units of numerous apolipoproteins and have been described as being responsible for the interaction of apolipoproteins with phospholipids in high-density lipoproteins (HDL). Furthermore. apolipoproteins. and especially apolipoprotein Al (apoAl), are involved in various biological functions of these circulating particles in plasma. Studies with synthetic peptides corresponding to domains of the apoAl sequence have however shown that short 39-residue fragments do not interact strongly enough with phospholipids to generate particles that correctly mimic the physicochemical properties of HDL reconstituted with native apoAl [Vanloo, B., Demoor, L., Boutil- Ion. C.. Lins, L.. Baert, J,, Fruchart, J. C.. Tartar. A. & Rosseneu, M. (1995) Association of synthetic peptide fragments of human apolipoprotein A-I with phospholipids. i. Lipid Res. 36. 1686-1696.1. Here we show that synthetic branched multirneric peptides. often used as carriers for the design of synthetic vaccines (multiple-antigen peptides). can be used to mimic the physicochemical properties of apoAl in HDL. This type of molecule is obtained by using a small core matrix of Lys residues bearing radially branched synthetic peptides as dendritic arms. We compared the lipid-binding capacities and the structural properties of a linear peptide corresponding to residues 145--183 of apoAl [apoAl-(145-183)-peptide) with those of two multimeric peptides consisting respectively of three [trimeric apoAl-(145-183)j and four copies [tetrameric apoAl-( 145- 183)] of the selected sequence. branched on a covalent core matrix. This paper provides evidence for the increased abilities of the multimeric peptides to associate with phospholipids compared with the short linear peptides. Moreover, the trimeric apoAl-(145 - 183) peptide was most efficient in mimicking the physicochemical and structural properties of native apoAl in reconstituted HDL. As tools adequate to unravel the structure/function relationship of separate apolipoprotein domains are still missing. these multirneric peptides might constitute an alternative approach to linear peptides which are poor mimetics and to protein mutants which are difficult to produce and only provide information about the total sequence. [ABSTRACT FROM AUTHOR]

The 154-171 segment of the human Lecithin-cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment and its orientation at a lipid/water interface are similar to those of some of the helical repeats of apolipoprotein A-IV and E. This domain was therefore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis we synthesized the LCAT-(154-171)-peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydrophobicity through Y1561, L1601, L163I and Y171W substitutions. The structural and lipid-binding properties of these synthetic peptides were investigated by turbidity, fluorescence, electron microscopy and circular dichroism. The wild-type peptide, the R158C variant in its dimeric form, as well as the more hydrophobic peptide, associated with phospholipids, whereas the Y156N and the R158C variant in its monomeric form did not. However, only the complexes generated with the hydrophobic variant were stable enough to resist dissociation during gel filtration. The wild-type peptide and hydrophobic variant formed discoidal complexes with dimyristoylglycerophosphocholine (Myr2GroPCho) as shown by negative staining electron microscopy. Comparison of the properties of the wild-type and hydrophobic variant LCAT-(154171)-peptide stresses the contribution of the hydrophobic face of the amphipathic helix to the formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the YI56N variant peptide for lipids, as this mutation decreased the mean hydrophobicity of the hydrophobic face of the amphipathic helix. These results support the hypothesis that the 154-171 segment of LCAT might be involved in the interaction of the enzyme with its lipid substrate and suggest that the decreased activity of the Y156N natural LCAT mutant might result from a decreased affinity of this mutant for lipids. [ABSTRACT FROM AUTHOR]

We purified an antimicrobial protein of 76 residues, denoted bovine antimicrobial protein-1 (BAMP-1), from fetal calf serum using hydrophobic chromatography, gel filtration, and reverse-phase high-performance liquid chromatography. The amino acid sequence of BAMP-1 was similar to that of human apolipoprotein A-II (apo A-II), a major component of high-density lipoprotein (HDL), and the amino acid composition was almost identical to that of a previously reported candidate for bovine apo A-II. BAMP-1 was recovered from the post-HDL fraction, but not from the HDL fraction of the serum and was associated with a small amount of triglycerides (5%, w/w). These results suggest that BAMP-1 is the bovine homologue of apo A-II and is present in almost free form in serum. BAMP-1 showed a weak growth-inhibitory activity against Escherichia coli and yeasts tested in phosphate-buffered saline (PBS). [ABSTRACT FROM AUTHOR]

Two-dimensional NMR, circular dichroism (CD) experiments and molecular modeling were performed to study the secondary structure of a 44-mer peptide fragment derived from the C4 region of gp120 of human immunodeficiency virus in aqueous solution. It was found a nascent helical structure exists following a type I turn near the N-terminus of the peptide. The proline residue in the turn appears to serve as a helix initiator. The helical structure was in fast dynamic equilibrium with β- or random coil form on the NMR scale. A reverse turn was identified at a section containing two consecutive proline residues. A nascent helical structure has been detected for the region near the C-terminus of the 44-mer peptide. Higher helical content for the peptide is also indicated by CD studies on TFE titration. Thus it is proposed that, in more apolar medium, the Pro-Pro turn and the segment amino-terminal to it, spanning about 20 amino acids, may be converted into helix structure. Moreover, the region near the C-terminus of the peptide may also be induced into helix, so that a helix-turn-helix structure may be formed in the C4 domain of gp120. A helical wheel representation of this stretch shows amphipathicity of the helix. The biological implication of the conformational adaptibility of the peptide was discussed. © Munksgaard 1997. [ABSTRACT FROM AUTHOR]

Magainin2 is a 23-residue antibiotic peptide that disrupts the ionic gradient across certain cellmembranes. Two-dimensional 1H NMR spectroscopy was used to investigate the structure ofthe peptide in three of the membrane environments most commonly employed in biophysicalstudies. Sequence-specific resonance assignments were determined for the peptide inperdeuterated dodecylphosphocholine (DPC) and sodium dodecylsulfate micelles andconfirmed for the peptide in 2,2,2-trifluoroethanol solution. The secondary structure is shownto be helical in all of the solvent systems. The NMR data were used as a set of restraints fora simulated annealing protocol that generated a family of three-dimensional structures of thepeptide in DPC micelles, which superimposed best between residues 4 and 20. For theseresidues, the mean pairwise rms difference for the backbone atoms is 0.47 ± 0.10Å from the average structure. The calculated peptide structures appear to be curved,with the bend centered at residues Phe12 and Gly13. [ABSTRACT FROM AUTHOR]

Anoplin is a linear 10-amino acid amphipathic peptide (Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu- NH 2 ) derived from the venom sac of the solitary wasp. It has broad antimicrobial activity, including an antibacterial one. However, the inhibition of bacterial growth requires several dozen micromolar concentrations of this peptide. Anoplin is positively charged and directly interacts with anionic biological membranes forming an α-helix that disrupts the lipid bilayer. To improve the bactericidal properties of anoplin by stabilizing its helical structure, we designed and synthesized its analogs with hydrocarbon staples. The staple was introduced at two locations resulting in different charges and amphipathicity of the analogs. Circular dichroism studies showed that all modified anoplins adopted an α-helical conformation, both in the buffer and in the presence of membrane mimics. As the helicity of the stapled anoplins increased, their stability in trypsin solution improved. Using the propidium iodide uptake assay in Escherichia coli and Staphylococcus aureus , we confirmed the bacterial membrane disruption by the stapled anoplins. Next, we tested the antimicrobial activity of peptides on a range of Gram-negative and Gram-positive bacteria. Finally, we evaluated peptide hemolytic activity on sheep erythrocytes and cytotoxicity on human embryonic kidney 293 cells. All analogs showed higher antimicrobial activity than unmodified anoplin. Depending on the position of the staple, the peptides were more effective either against Gram-negative or Gram-positive bacteria. Anoplin[5-9], with a lower positive charge and increased hydrophobicity, had higher activity against Gram-positive bacteria but also showed hemolytic and destructive effects on eukaryotic cells. Contrary, anoplin[2-6] with a similar charge and amphipathicity as natural anoplin effectively killed Gram-negative bacteria, also pathogenic drug-resistant strains, without being hemolytic and toxic to eukaryotic cells. Our results showed that anoplin charge, amphipathicity, and location of hydrophobic residues affect the peptide destructive activity on the cell wall, and thus, its antibacterial activity. This means that by manipulating the charge and position of the staple in the sequence, one can manipulate the antimicrobial activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wojciechowska, Macyszyn, Miszkiewicz, Grzela and Trylska.)

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein E rns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant E rns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form E rns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of E rns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences. [ABSTRACT FROM AUTHOR]

• Atg16 in the budding yeast S. cerevisiae is an intrinsically disordered polypeptide. • The C terminal amphipathic helix (at amino acid residues 113–131) anchors Atg16 on membranes. • The coiled-coil domain of Atg16 (at amino acid residues 64–99) mediates homodimerization in vivo. • Both membrane binding and homodimerization are required for Atg16 function in autophagy. Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy. [ABSTRACT FROM AUTHOR]

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)