Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins

Cystine-knot miniproteins are members of a large family of small proteins that are defined by a common structural scaffold which is stabilized by three intramolecular disulfide bonds. Cystine-knot miniproteins display a broad spectrum of therapeutically useful natural biological activities and several family members are marketed as therapeutics or are in clinical development. Because of their extraordinary intrinsic chemical and proteolytic stability they provide promising scaffolds for the introduction of therapeutically relevant functionalities. Several successful engineering efforts have been reported to generate miniproteins with novel activities by rational design via functional loop grafting or by directed evolution via screening of scaffold-constrained random libraries. Owing to their small size they are amenable to recombinant as well as to chemical routes of synthesis, which opens up new avenues in optimizing biological activity, specificity and bioavailability by site-specific modification, introduction of non-natural amino acids or chemical conjugation.     

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.     

Uncovering small membrane proteins in pathogenic bacteria: Regulatory functions and therapeutic potential

Bacterial small proteins (below 50 amino acids) encoded by small open reading frames (sORFs) are recognized as an emerging class of functional molecules that have been largely overlooked in the past. While some were uncovered serendipitously, global approaches have recently been developed to detect these sORFs. A large portion of small proteins appears to be hydrophobic and located in the bacterial membrane. In the present review, we describe functional small hydrophobic proteins discovered in pathogenic bacteria and report recent advances in the discovery of additional ones. Small membrane proteins contribute to bacterial adaptation to changing environments and often appear to be implicated in negative feedback regulation loops by modulating the function or stability of larger membrane proteins. A subset of these proteins belongs to toxin-antitoxin modules. We highlight the features of characterized hydrophobic small proteins that may pave the way for identification of the functional small proteins among novel sORFs discovered. Besides providing new insights into bacterial pathogenesis, identification of naturally occurring small hydrophobic proteins of pathogenic bacteria can lead to new therapeutic interventions, as recently shown with the development of synthetic peptides derived from natural small proteins that display antibacterial or antivirulence properties.     

RAN proteins in neurodegenerative disease: Repeating themes and unifying therapeutic strategies

Microsatellite-expansion mutations cause >50 neurological diseases but there are no effective treatments. Mechanistic studies have historically focused on protein loss-of-function and protein or RNA gain-of-function effects. It is now clear that many expansion mutations are bidirectionally transcribed producing two toxic expansion RNAs, which can produce up to six mutant proteins by repeat associated non-AUG (RAN) translation. Multiple types of RAN proteins have been shown to be toxic in cell and animal models, to lead to common types of neuropathological changes, and to dysregulate key pathways. How RAN proteins are produced without the canonical AUG or close-cognate AUG-like initiation codons is not yet completely understood but RNA structure, flanking sequences and stress pathways have been shown to be important. Here, we summarize recent progress in understanding the role of RAN proteins, mechanistic insights into their production, and the identification of novel therapeutic strategies that may be applicable across these neurodegenerative disorders.     

Exosomes and microRNAs: New potential therapeutic candidates in Alzheimer disease therapy

Exosomes are biological nanocarriers which could be involved in a variety of basic physiological events. They exert their effects via targeting their cargos (i.e., DNAs, messenger RNAs, microRNAs [miRNAs], and proteins) to host cells, which led to change behaviors of recipient cells. One of the important aspects of exosomes is the roles of them in disease conditions. Increasing evidence indicated that exosomes are one of the main players in Alzheimer’s disease (AD) pathogenesis. Hence, it seems that these nanocarriers could be used as diagnostic and therapeutic biomarkers in AD treatment. Another important player in AD pathogenesis is miRNA. MiRNAs are short noncoding RNAs which exert their effects as epigenetic regulators. These molecules involved in different stages of AD. Therefore, miRNAs could be used as prognostic, diagnostic, and therapeutic biomarkers in AD. Here, we summarized various roles of exosomes and application of them in AD pathogenesis. Moreover, we highlighted the utilization of miRNAs as a therapeutic option in AD therapy.     

Autosomal dominant polycystic kidney disease: Disrupted pathways and potential therapeutic interventions

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that occurs in different races worldwide. It is characterized by the development of a multitude of renal cysts, which leads to massive enlargement of the kidney and often to renal failure in adulthood. ADPKD is caused by a mutation in PKD1 or PKD2 genes encoding the proteins polycystin-1 and polycystin-2, respectively. Recent studies showed that cyst formation and growth result from deregulation of multiple cellular pathways like proliferation, apoptosis, metabolic processes, cell polarity, and immune defense. In ADPKD, intracellular cyclic adenosine monophosphate (cAMP) promotes cyst enlargement by stimulating cell proliferation and transepithelial fluid secretion. Several interventions affecting many of these defective signaling pathways have been effective in animal models and some are currently being tested in clinical trials. Moreover, the stem cell therapy can improve nephropathies and according to studies were done in this field, can be considered as a hopeful therapeutic approach in future for PKD. This study provides an in-depth review of the relevant molecular pathways associated with the pathogenesis of ADPKD and their implications in development of potential therapeutic strategies.