What is the role of thermodynamics on protein stability?

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article.     

Has the code for protein translocation been broken?

Polypeptides chains are segregated by the translocon channel into secreted or membrane-inserted proteins. Recent reports claim that an in vivo system has been used to break the "amino acid code" used by translocons to make the determination of protein type (i.e. secreted or membrane-inserted). However, the experimental setup used in these studies could have confused the derivation of this code, in particular for polar amino acids. These residues are likely to undergo stabilizing interactions with other protein components in the experiment, shielding them from direct contact with the inhospitable membrane. Hence, it is our view that the "code" for protein translocation has not yet been deciphered and that further experiments are required for teasing apart the various energetic factors contributing to protein translocation.     

What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy?

A growing body of evidence shows that mitochondria are heterogeneous in terms of structure and function. Increased heterogeneity has been demonstrated in a number of disease models including ischemia-reperfusion and nutrient-induced beta cell dysfunction and diabetes. Subcellular location and proximity to other organelles, as well as uneven distribution of respiratory components have been considered as the main contributors to the basal level of heterogeneity. Recent studies point to mitochondrial dynamics and autophagy as major regulators of mitochondrial heterogeneity. While mitochondrial fusion mixes the content of the mitochondrial network, fission dissects the mitochondrial network and generates depolarized segments. These depolarized mitochondria are segregated from the networking population, forming a pre-autophagic pool contributing to heterogeneity. The capacity of a network to yield a depolarized daughter mitochondrion by a fission event is fundamental to the generation of heterogeneity. Several studies and data presented here provide a potential explanation, suggesting that protein and membranous structures are unevenly distributed within the individual mitochondrion and that inner membrane components do not mix during a fusion event to the same extent as the matrix components do. In conclusion, mitochondrial subcellular heterogeneity is a reflection of the mitochondrial lifecycle that involves frequent fusion events in which components may be unevenly mixed and followed by fission events generating disparate daughter mitochondria, some of which may fuse again, others will remain solitary and join a pre-autophagic pool.     

Yeast lipid rafts? – An emerging view

In various eukaryotes, sterol-rich membrane domains have been proposed to play an important role in polarization and compartmentalization of the plasma membrane. Several studies have reported the cellular distribution of sterols in genetically tractable yeast species and the identification of molecules that might regulate the localization of sterol-rich membrane domains. Here, we attempt to synthesize our understanding of the function and organization of these domains from the study of fungi and identify some outstanding issues.     

All G protein βγ complexes are capable of translocation on receptor activation

Heterotrimeric G proteins transduce signals sensed by transmembrane G protein coupled receptors (GPCRs). A subfamily of G protein βγ subunit types has been shown to selectively translocate from the plasma membrane to internal membranes on receptor activation. Using 4D imaging we show here that Gβγ translocation is not restricted to some subunit types but rather all 12 members of the family of mammalian γ subunits are capable of supporting βγ translocation. Translocation kinetics varies widely depending on the specific γ subunit type, with t(1/2) ranging from 10s to many minutes. Using fluorescence complementation, we show that the β and γ subunits translocate as βγ dimers with kinetics determined by the γ subunit type. The expression patterns of endogenous γ subunit types in HeLa cells, hippocampal neurons and cardiomyocytes are distinctly different. Consistent with these differences, the βγ translocation rates vary widely. βγ translocation rates exhibit the same γ subunit dependent trends regardless of the specific receptor type or cell type showing that the translocation rates are intrinsic to the γ subunit types. βγ complexes with widely different rates of translocation had differential effects on muscarinic stimulation of GIRK channel activity. These results show that G protein βγ translocation is a general response to activation of GPCRs and may play a role in regulating signaling activity.     

AMP-activated protein kinase - An archetypal protein kinase cascade?

Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.     

What is the protein design alphabet?

Selecting a protein sequence that corresponds to a specific three-dimensional protein structure is known as the protein design problem. One principal bottleneck in solving this problem is our lack of knowledge of precise atomic interactions. Using a simple model of amino acid interactions, we determine three crucial factors that are important for solving the protein design problem. Among these factors is the protein alphabet-a set of sequence elements that encodes protein structure. Our model predicts that alphabet size is independent of protein length, suggesting the possibility of designing a protein of arbitrary length with the natural protein alphabet. We also find that protein alphabet size is governed by protein structural properties and the energetic properties of the protein alphabet units. We discover that the usage of average types of amino acid in proteins is less than expected if amino acids were chosen randomly with naturally occurring frequencies. We propose three possible scenarios that account for amino acid underusage in proteins. These scenarios suggest the possibility that amino acids themselves might not constitute the alphabet of natural proteins.     

Obesogens and obesity—An alternative view?

It is accepted that diet is a major contributor to the obesity epidemic, but environmental ‘obesogenic’ chemicals have also been suggested recently as playing a role, based on in vitro, animal and epidemiological studies. Using two such ‘obesogen’ examples (bisphenol A, certain phthalate esters), we argue that their association with obesity and obesity‐related disorders in humans could be circumstantial, and thus non‐causal, because a Western style diet increases exposure to these compounds. This possibility needs to be addressed before further (confounded) epidemiological studies on ‘obesogens’ are undertaken.     

Hypoxic preconditioning promotes the translocation of protein kinase C ? binding with caveolin-3 at cell membrane not mitochondrial in rat heart

Protein kinase C has been shown to play a central role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae are critical for PKC signaling, we sought to determine whether hypoxic preconditioning promotes translocation and association of PKC isoforms with caveolin-3. A cellular model of hypoxic preconditioning from adult rat cardiac myocytes (ARCM) or H9c2 cells was employed to examine PKC isoforms by molecular, biochemical and cellular imaging analysis. Hypoxia was induced by incubating the cells in an airtight chamber in which O₂ was replaced by N₂ with glucose-free Tyrode''s solution. Cells were subjected to hypoxic preconditioning with 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Western blot data indicated that the band intensity for PKCϵ, PKCδ or PKCα, but not PKCβ and PKCζ was enhanced significantly by hypoxic preconditioning from the caveolin-enriched plasma membrane interactions. Immunoprecipitation experiments from the caveolin-enriched membrane fractions of ARCM showed that the level of PKCϵ, PKCδ and PKCα in the anti-caveolin-3 immunoprecipitates was also increased by hypoxic preconditioning. Further, our FRET analysis in H9c2 cells suggested that there is a minimum FRET signal for caveolin-3 and PKCϵ along cell peripherals, but hypoxic preconditioning enhanced the FRET signal, indicating a potential interaction between caveolin-3 and PKCϵ. And also treatment of the cells with hypoxic preconditioning led to a smaller amount of translocation of PKCϵ to the mitochondria than that to the membrane. We demonstrate that hypoxic preconditioning promotes rapid association of PKCϵ, PKCδ and PKCα with the caveolin-enriched plasma membrane microdomain of cardiac myocytes, and PKCϵ via direct molecular interaction with caveolin-3. This regulatory mechanism may play an important role in cardioprotection.